Gene fusions and gene variants associated with cancer

ABSTRACT

The disclosure provides gene fusions, gene variants, and novel associations with disease states, as well as kits, probes, and methods of using the same.

RELATED APPLICATIONS

This application is a divisional application under 35 U.S.C. § 120 of allowed U.S. application Ser. No. 14/672,066, filed Mar. 27, 2015, which application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/092,898, filed Dec. 17, 2014; and U.S. Provisional Application No. 62/004,727, filed May 29, 2014; and U.S. Provisional Application No. 61/993,732, filed May 15, 2014; and U.S. Provisional Application No. 61/971,455, filed Mar. 27, 2014; and U.S. application Ser. No. 14/672,066 is a Continuation-in-Part of U.S. application Ser. No. 14/214,300, filed Mar. 14, 2014, now abandoned, which application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 61/940,226, filed Feb. 14, 2014; and U.S. Provisional Application No. 61/935,650, filed Feb. 4, 2014; and U.S. Provisional Application No. 61/915,392, filed Dec. 12, 2013; and U.S. Provisional Application No. 61/907,939, filed Nov. 22, 2013; and U.S. Provisional Application No. 61/860,115, filed Jul. 30, 2013; and U.S. Provisional Application No. 61/824,253, filed May 16, 2013; and U.S. Provisional Application No. 61/813,465, filed Apr. 18, 2013; and U.S. Provisional Application No. 61/813,182 filed Apr. 17, 2013. The entire contents of the aforementioned applications are incorporated by reference herein.

SEQUENCE LISTING

This application hereby incorporates by reference the material of the electronic Sequence Listing filed concurrently herewith. The material in the electronic Sequence Listing is submitted as a text (.txt) file entitled “LT00802_1_2ST25.txt” created on Mar. 27, 2015, last modified Feb. 12, 2018 which has a file size of 103,558, and is herein incorporated by reference in its entirety.

FIELD OF INVENTION

The present invention relates generally to gene fusions and gene variants that are associated with cancer.

BACKGROUND

Aberrations such as chromosomal translocations and gene variants are frequently found in human cancer cells. Chromosomal translocations may result in a chimeric gene expressing a fusion transcript which is then translated into a fusion protein that affects normal regulatory pathways and stimulates cancer cell growth. Gene variants may also result in aberrant proteins that affect normal regulatory pathways.

The identification of new fusion genes, new variants of known fusion genes, and gene variants or alleles provides an opportunity for additional diagnostics and cancer treatment targets.

BRIEF SUMMARY OF THE INVENTION

The disclosure provides novel gene fusion variants and gene fusion-disease state associations. The gene fusions provided herein are associated with certain cancers. The disclosure further provides probes, such as amplification primer sets and detection probes, as well as methods and systems of detection, diagnosis, and treatment and kits that include or detect the gene fusions disclosed herein.

In certain embodiments, novel mutations and fusions associated with cancer are disclosed in Tables 41-44.

The disclosure further provides reaction mixtures, sets of probes, kits, methods, isolated nucleic acids comprising or to detect the novel mutations and fusion events of Tables 41-44.

In one embodiment, the disclosure provides a reaction mixture comprising a probe or a set of probes that specifically recognize a gene fusion selected from Table 1-Table 3, Table 19, and Table 22. The set of probes can be, for example a set of amplification primers. In another embodiment, provided herein is a reaction mixture that includes a set of primers that flank a gene fusion selected from Table 1-Table 3, Table 19, and Table 22 in a target nucleic acid. For example, the set of primers can each bind to a target sequence in the human genome within 1000, 750, 500, 250, 100, 90, 80, 75, 70, 65, 50, or 25 nucleotides of opposite sides of the one of the fusion breakpoints identified in Tables 4-6, 20, and 23. The reaction mixture of this embodiment can further include a detector probe that binds to either side of a breakpoint in a gene fusion selected from Table 1-Table 3, Table 19, and Table 22, or that binds a binding region that spans the breakpoint in a gene fusion selected from Table 1-Table 3, Table 19, and Table 22, including specific embodiments where the breakpoint is identified in Tables 4-6, 20, and 23. In exemplary embodiments, the detector probe binds to a target sequence in the human genome within 1000, 750, 500, 250, 100, 90, 80, 75, 70, 60, 50, or 25 nucleotides of one of the fusion breakpoints identified in Tables 4-6, 20, and 23. The reaction mixture that includes a detector probe, or does not include a detector probe, can further include a polymerase, a reverse transcriptase, dNTPs, and/or a uracil DNA deglycosylase (UDG). The polymerase, the reverse transcriptase, and the UDG are typically not from human origin. The polymerase in illustrative embodiments is a thermostable polymerase such as a Taq polymerase. In certain embodiments, the dNTPs in the reaction mixture include dUTP, and the reaction mixture can in certain examples, be devoid of dTTP. Furthermore, the reaction mixture can include an amplicon, such as a DNA amplicon that includes one or more deoxyuridine (“dU”) residues. In certain embodiments the reaction mixture includes a DNA amplicon that includes one or more dU residues for every deoxythymidine residue in the corresponding human genomic sequence. In certain embodiments, the amplicon includes a segment for which a corresponding sequence is not found in the human genome, such as, for example, a DNA barcode sequence. The non-human segment can be for example, 5-10,000, 5-5000, 5-1000, 5-500, 5-100, 5-50, 5-25, 5-10, 10-10,000, 10-5000, 10-1000, 10-500, 10-100, 10-50, or 10-25 nucleotides in length. In certain embodiments, the amplicon includes segment that corresponds to the region of the human genome that spans an intron, but the amplicon does not include a segment corresponding to the intron. The reaction mixture can further include a target nucleic acid, for example a human target nucleic acid. The human target nucleic acid can be, for example, isolated from a biological sample from a person suspected of having a cancer selected from: BLCA=bladder carcinoma, BRCA=breast carcinoma, CESC=cervical cell carcinoma, COAD=colon adenocarcinoma, GBM=glioblastoma multiforme, HNSC=head and neck squamous cell carcinoma, KIRK=clear cell renal cell carcinoma, KIRP=kidney renal papillary cell carcinoma, LAML=acute myeloid leukemia, LGG=brain lower grade glioma, LIHC=liver hepatocellular carcinoma, LUAD=lung adenocarcinoma, LUSC=squamous cell lung carcinoma, OV=ovarian serous adenocarcinoma, PRAD=prostate adenocarcinoma, READ=rectal adenocarcinoma, SKCM=cutaneous melanoma, STAD=stomach adenocarcinoma, THCA=thyroid carcinoma, and UCEC=uterine corpus endometrioid carcinoma. In certain embodiments, the target nucleic acid is from a tumor, for example a tumor of one of the cancer types listed in the preceding sentence.

In another embodiment, a set of probes that specifically recognizes a nucleic acid comprising at least one of SEQ ID NOs: 1-289 (gene fusions) is provided. In another embodiment, provided herein is a set of primers that specifically amplify a target nucleic acid that includes at least 25, 30, 40, 50, 75, 100, 125, 150, 200, or all of SEQ ID NOs: 1-289, or that amplifies up to 25, 30, 40, 50, 75, 100, 125, 150, 200, or all of SEQ ID NOs: 1-289. In another embodiment, provided herein is a qPCR assay, such as a TaqMan™ assay or a Molecular Beacons™ assay, that specifically amplifies and detects a target nucleic acid that includes at least 25, 30, 40, 50, 75, 100, 125, 150, 200, or all of SEQ ID NOs: 1-289.

The disclosure also provides an isolated nucleic acid comprising at least one sequence selected from a segment that includes at least 25, 30, 40, 50, 75, 100, 125, 150 200, or all of SEQ ID NOs: 1-289 or that includes up to 25, 30, 40, 50, 75, 100, 125, 150, 200, or all of SEQ ID NOs: 1-289. The isolated nucleic acid can include a first primer on a 5′ end. Furthermore, the nucleic acid can be single stranded or double stranded. In certain embodiments, the isolated nucleic acid includes a segment for which a corresponding sequence is not found in the human genome, such as, for example, a DNA barcode sequence. The segment can be for example, 5-10,000, 5-5000, 5-1000, 5-500, 5-100, 5-50, 5-25, 5-10, 10-10,000, 10-5000, 10-1000, 10-500, 10-100, 10-50, or 10-25 nucleotides in length.

The disclosure, in other embodiments, provides a kit that includes a detector probe and/or a set of probes, for example, a set of amplification primers, that specifically recognize a nucleic acid comprising a breakpoint for a gene fusion selected from Table 1-Table 3, Table 19, and Table 22. For example, in certain embodiments the detector probe or set of amplification primers are designed to amplify and/or detect a nucleic acid that includes up to 25, 30, 40, 50, 75, 100, 125, 150, 200, or all of at least one of SEQ ID NOs: 1-289. The kit can further include, in one or more separate or in the same vessel, at least one component from an amplification reaction mixture, such as a polymerase, dNTPs, a reverse transcriptase, and/or UDG, typically the reverse transcriptase, polymerase and UDG are not from human origin. In certain embodiments, the dNTPs include dUTP, and in illustrative examples are devoid of dTTP. The polymerase in illustrative embodiments is a thermostable polymerase such as a Taq polymerase. Furthermore, the kit can include a control nucleic acid. For example the control nucleic acid can include a sequence that includes the break point in a gene fusion selected from Table 1-Table 3, Table 19, and Table 22, such as a nucleic acid that includes at least 25, 30, 40, 50, 75, 100, 125, 150, 200, or all of SEQ ID NOs: 1-289 or a nucleic acid that includes up to 25, 30, 40, 50, 75, 100, 125, 150, 200, or all of SEQ ID NOs: 1-289.

A method of detecting a cancer is provided comprising amplifying a nucleic acid that spans a breakpoint in a gene fusion selected from Table 1-Table 3, Table 19, and Table 22, for example the nucleic can include a sequence selected from SEQ ID NOs: 1-289, and detecting the presence of the nucleic acid, wherein the presence of the nucleic acid indicates a cancer is present in the sample. In another method, provided herein is a method of detecting a cancer selected from, bladder, colon, breast, endometrial, melanoma, ovarian, glioblastoma, glioma, leukemia, renal cell carcinoma, thyroid, and prostate adenocarcinoma that includes generating an amplicon that includes a sequence selected from SEQ ID NOs: 1-289 and detecting the presence of the amplicon, wherein the presence of the amplicon indicates bladder, colon, melanoma, ovarian, glioblastoma, lung, glioma, leukemia, renal cell carcinoma, thyroid, endometrial endometrioid adenocarcinoma, breast and prostate adenocarcinoma is present in the sample. The amplicon typically includes primers that were extended to form the amplicon. The cancer is selected from bladder urothelial carcinoma, breast carcinoma, endometrial endometrioid adenocarcinoma, colon adenocarcinoma, glioblastoma multiforme, clear cell renal cell carcinoma, papillary renal cell carcinoma, acute myeloid leukemia, brain lower grade glioma, lung adenocarcinoma, ovarian serous cystadenocarcinoma, prostate adenocarcinoma, rectal cutaneous melanoma, and thyroid gland carcinoma. The amplicon that is generated, in certain illustrative embodiments is a DNA amplicon that includes dU residues, and in certain examples includes no dT residues. In the methods provided in this paragraph, the amplicon can be generated using reaction mixtures provided herein. In certain embodiments, the method includes detecting expression of a nucleic acid that spans a breakpoint in a gene fusion selected from Table 1-Table 3, Table 19, and Table 22. Methods for detecting expression typically include a step of isolating RNA from a sample, such as a tumor sample, which can be a formalin fixed sample in illustrative embodiments.

In one embodiment, the reaction mixture includes a dye selected from SYBR Green, SBYR Greener, Fluorescein, Oregon Green, FAM, TET, JOE, VIC, Yakima Yellow, HEX, Cy3, Bodipy TMR, NED, TAMRA, Cy3.5, ROX, Texas Red, LightCycler Red, Bodipy 630/650, Alexa Fluor 647, Cy5, Alexa Fluor 660, or Cy 5.5. In certain embodiments, the dye is attached to a detably-labeled probe in the reaction mixture. In other embodiments, the dye is bound to the amplicon directly or through a detectably-labeled probe.

A kit comprising a probe or a set of probes, for example, a detectable probe or a set of amplification primers that specifically recognize a nucleic acid comprising a break point from Tables 4-6, 20, and 23 is provided. The kit can further include, in the same vessel, or in certain preferred embodiments, in a separate vessel, a component from an amplification reaction mixture, such as a polymerase, typically not from human origin, dNTPs, and/or UDG. Furthermore, the kit can include a control nucleic acid. For example the control nucleic acid can include a sequence that includes a break point selected from Tables 4-6, 20, and 23.

In another embodiment, provided herein a gene fusion that includes the gene fusions identified in Tables 1-3, 19, and 22. In illustrative embodiments, the gene fusions include one of the breakpoints identified in Tables 4-6, 20, and 23. Accordingly, provided herein is an isolated gene fusion nucleic acid of between 100 and 10,000 nucleotides in length and comprising at least 25 nucleotides on either side of one of the break points in Tables 4-6, 20, and 23.

In a related embodiment, provided herein is an isolated gene fusion nucleic acid comprising at least one of the break points in Tables 4-6, 20, and 23. In certain embodiments, the isolated gene fusion nucleic acid comprises at least 25, 30, 40, 50, 75, 100, 125, 150, 200, or all of SEQ ID NOs: 1-289 or a nucleic acid that includes up to 25, 30, 40, 50, 75, 100, 125, 150, 200, or all of SEQ ID NOs: 1-289. The isolated gene fusion nucleic acid can have a length, for example, of between 50 and 100,000 nucleotides, between 100 and 50,000 nucleotides, between 100 and 25,000 nucleotides, between 100 and 10,000 nucleotides, between 100 and 5,000 nucleotides, between 100 and 2500 nucleotides, between 100 and 1,000 nucleotides, between 100 and 500 nucleotides, between 100 and 250 nucleotides, between 100 and 200 nucleotides, between 250 and 10,000 nucleotides, between 250 and 5,000 nucleotides, between 250 and 1,000 nucleotides, or between 250 and 500 nucleotides. In certain aspects, the isolated gene fusion nucleic acid is DNA. In certain illustrative embodiments, the isolated nucleic gene fusion is devoid of intron sequences but spans a region that in the genome includes one or more introns. In certain embodiments, the isolated gene fusion nucleic acid is a cDNA.

In another embodiment, an isolated gene fusion nucleic acid is provided comprising at least one of the break points in Tables 4-6, 20, and 23.

In another embodiment is a method to detect a cancer selected from bladder urothelial carcinoma, breast carcinoma, endometrial endometrioid adenocarcinoma, colon adenocarcinoma, glioblastoma multiforme, clear cell renal cell carcinoma, papillary renal cell carcinoma, acute myeloid leukemia, brain lower grade glioma, lung adenocarcinoma, ovarian serous cystadenocarcinoma, prostate adenocarcinoma, rectal cutaneous melanoma, and thyroid gland carcinoma in a sample by detecting the presence of a gene fusion selected from Table 1-Table 3, Table 19, and Table 22.

The disclosure provides novel gene variants and gene variant-disease state associations. The gene variants can have one or more mutations that result in a variant protein. The gene variants provided herein are associated with certain cancers. The gene variants result in protein variants. The disclosure further provides probes, such as amplification primer sets and detection probes, as well as methods of detection, diagnosis, and treatment and kits that include or detect the gene variants disclosed herein.

In one embodiment, the disclosure provides a composition and a kit comprising a set of probes that specifically recognize the nucleotide sequence that encodes a gene variant selected from Table 7 and/or Table 11. The set of probes can be, for example a set of amplification primers. In another embodiment, provided herein is a composition that includes a set of primers that flank a gene variant that encodes one or more variants in Table 7 and/or Table 11. The reaction mixture of this embodiment can further include a detector probe that binds to a nucleotide sequence including a gene variant selected from Table 7 and/or Table 11. The reaction mixture that includes a detector probe or does not include a detector probe, can further include a polymerase, dNTPs, and/or a uracil DNA deglycosylase (UDG). The polymerase and UDG are typically not from a human origin. The reaction mixture can further include a target nucleic acid, for example a human target nucleic acid. The human target nucleic acid can be, for example, isolated from a biological sample from a person suspected of having a cancer. The cancer can be selected from: BLCA=bladder carcinoma, BRCA=breast carcinoma, CESC=cervical cell carcinoma, COAD=colon adenocarcinoma, GBM=glioblastoma multiforme, HNSC=head and neck squamous cell carcinoma, KIRK=clear cell renal cell carcinoma, KIRP=kidney renal papillary cell carcinoma, LAML=acute myeloid leukemia, LGG=brain lower grade glioma, LIHC=liver hepatocellular carcinoma, LUAD=lung adenocarcinoma, LUSC=squamous cell lung carcinoma, OV=ovarian serous adenocarcinoma, PRAD=prostate adenocarcinoma, READ=rectal adenocarcinoma, SKCM=cutaneous melanoma, STAD=stomach adenocarcinoma, THCA=thyroid carcinoma, and UCEC=uterine corpus endometrioid carcinoma.

The nucleotide sequence that encodes one or more gene variants in Table 7 and/or Table 11 can be any size that encompasses the variation. For example, the nucleotide sequence can be any size that can be easily copied using a primer and/or detected using a probe.

In another embodiment, a set of probes that specifically recognize a nucleic acid coding for a gene variant selected from Table 7 and/or Table 11 (gene variants) is provided. In another embodiment, provided herein is a set of primers that specifically amplify a target nucleic acid that codes for a gene variant selected from Table 7 and/or Table 11. In another embodiment, provided herein is a qPCR assay, such as, but not limited to, a TaqMan™ assay, a Scorpions assay, or a Molecular Beacons™ assay that specifically amplifies and detects a target nucleic acid that codes for a gene variant selected from Table 7 and/or Table 11.

The disclosure also provides an isolated nucleic acid comprising at least one sequence that codes for one or more gene variants selected from Table 7 and/or Table 11. The isolated nucleic acid can include a first primer on a 5′ end. Furthermore, the nucleic acid can be single stranded or double stranded.

The disclosure, in other embodiments, provides a kit that includes a detector probe and/or a set of probes, for example, a set of amplification primers that specifically recognize a nucleic acid that codes for a gene variant selected from Table 7 and/or Table 11. For example, in certain embodiments the detector probe or set of amplification primers are designed to amplify and/or detect a nucleic acid that codes for a variant in Table 7 and/or Table 11. The kit can further include, in a separate or in the same vessel, a component from an amplification reaction mixture, such as a polymerase, typically not from human origin, dNTPs, and/or UDG. Furthermore, the kit can include a control nucleic acid. For example the control nucleic acid can include a sequence that includes the gene variant selected from Table 7 and/or Table 11.

A method of detecting a cancer is provided comprising amplifying a nucleic acid that encodes a gene variant selected from Table 7 and/or Table 11, for example the nucleic can include a sequence from one of the accession numbers in Table 7 and/or Table 11 except that the sequence contains the variant that codes for the gene variants in Table 7 and/or Table 11, and detecting the presence of the nucleic acid, wherein the presence of the nucleic acid indicates a cancer is present in the sample. In another method, provided herein is a method of detecting a cancer that includes generating an amplicon that includes a sequence encoding a variant selected from Table 7 and/or Table 11, and detecting the presence of the nucleic acid, wherein the presence of the nucleic acid indicates a cancer or cancer cell is present in the sample. The amplicon typically includes primers that are extended to form the amplicon. The cancer is selected from bladder carcinoma, breast carcinoma, cervical cell carcinoma, colon adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, clear cell renal cell carcinoma, kidney renal papillary cell carcinoma, acute myeloid leukemia, brain lower grade glioma, liver hepatocellular carcinoma, lung adenocarcinoma, squamous cell lung carcinoma, ovarian serous adenocarcinoma, prostate adenocarcinoma, rectal adenocarcinoma, cutaneous melanoma, stomach adenocarcinoma, thyroid carcinoma, and uterine corpus endometrioid carcinoma.

A kit comprising a set of probes, for example, a set of amplification primers that specifically recognize a nucleic acid comprising a gene variant from Table 7 and/or Table 11 is provided. The kit can further include, in a separate or in the same vessel, a component from an amplification reaction mixture, such as a polymerase, typically not from human origin, dNTPs, and/or UDG. Furthermore, the kit can include a control nucleic acid. For example the control nucleic acid can include a sequence that includes the gene variant from Table 7 and/or Table 11.

In certain embodiments, a set of probes that specifically recognize a nucleic acid comprising a gene variant from Table 7 and/or Table 11 is provided.

In another embodiment, a gene variant is provided comprising at least one of the gene variants in Table 7 and/or Table 11.

In another embodiment is a method to detect a cancer selected from bladder carcinoma, breast carcinoma, cervical cell carcinoma, colon adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, clear cell renal cell carcinoma, kidney renal papillary cell carcinoma, acute myeloid leukemia, brain lower grade glioma, liver hepatocellular carcinoma, lung adenocarcinoma, squamous cell lung carcinoma, ovarian serous adenocarcinoma, prostate adenocarcinoma, rectal adenocarcinoma, cutaneous melanoma, stomach adenocarcinoma, thyroid carcinoma, and uterine corpus endometrioid carcinoma in a sample by detecting the presence of a gene variant selected from Table 7 and/or Table 11. Gene variants can include, but are not limited to, ZNF479 variants R110, R295K, R295T, R2951, R3451, R345T, K438T, and T466K.

In another embodiment, a method of delivering a drug to a subject is provided, wherein the method includes detecting a genetic event identified in Table 15, and treating the subject with a drug, wherein the drug is believed to positively affect the clinical outcome of patients having the genetic event. In illustrative embodiments, the genetic event is associated with a gene found in Table 8 and the drug is listed in Table 8 as a companion for that gene. In another embodiment, provided herein is a method for determining if a subject receives a drug, the method includes detecting a genetic event identified in Table 15, and then delivering a drug to the subject if the detected genetic event is listed in Table 15 as associated with a poor prognosis, wherein the drug is believed to positively affect the clinical outcome of patients having the genetic event. In illustrative embodiments, the genetic event is associated with a gene found in Table 8 and the drug is listed in Table 8 as a companion for that gene.

In one embodiment, a kit is provided, wherein the kit comprises a set of probes, wherein each probe specifically hybridizes to a nucleic acid comprising a breakpoint from Tables 4-6, 20, and 23.

In one embodiment, a method is provided, the method comprising: amplifying a nucleic acid comprising at least one gene fusion from Tables 1-3, 19, and 22 from a sample; and detecting the presence of the at least one gene fusion by at least one of: contacting the composition with at least one probe, wherein each probe specifically hybridizes to the nucleic acid, or observing the presence of a non-natural or non-native chemical structure in the nucleic acid; wherein detecting the presence of the at least one gene fusion indicates that at least one cancer from Tables 1-3, 19, and 22 is present in the sample.

In one embodiment, a system is provided, the system comprising a nucleic acid amplifier configured to amplify a nucleic acid comprising at least one gene fusion from Tables 1-3, 19, and 22 from a sample, to yield an amplified nucleic acid; a detector configured to detect the presence of the at least one gene fusion in the amplified nucleic acid by at least one of (i) contacting the composition with at least one probe, wherein each probe specifically hybridizes to the nucleic acid, or (ii) observing the presence of a non-natural or non-native chemical structure in the nucleic acid, and further configured to transmit a detection indication; and a computer system configured to receive the detection indication and determine that at least one cancer from Tables 1-3, 19, and 22 is present in the sample, based on the detection indication.

In one embodiment, a non-transitory computer readable program storage unit is provided, the non-transitory computer readable program storage unit encoded with instructions that, when executed by a computer, perform a method, comprising receiving an input comprising at least a cancer type and an event type, wherein the cancer type is selected from Table 15 and the event type is selected from Table 15; querying a database for at least one entry comprising a plurality of fields, wherein the plurality of fields comprises at least one of the cancer type and the event type; and transmitting an output comprising at least one field of the plurality from the at least one entry, wherein the at least one field comprises at least one gene, at least one druggable gene, at least one drug targeting the at least one druggable gene, or a prognosis.

In one embodiment, a method is provided, wherein the method comprises administering to a patient having at least one gene fusion selected from the gene fusions listed in Tables 1-3, 19, and 22 at least one drug selected from the drugs listed in Tables 8, 16-17, 21, and 24.

In one embodiment, a method is provided, wherein the method comprises contacting a nucleic acid sample from a patient with a reaction mixture comprising a first primer complementary to a first gene and a second primer complementary to a second gene, wherein a fusion of the first gene and the second gene is detectable by the presence of an amplicon generated by the first primer and the second primer, wherein the fusion comprises a breakpoint selected from the breakpoints listed in Tables 4-6, 20, and 23.

In one embodiment, a non-transitory computer readable program storage unit is provided, the non-transitory computer readable program storage unit encoded with instructions that, when executed by a computer, perform a method, comprising receiving RNA sequence data from at least one cancer cell line; running at least one gene fusion caller on the sequence data, to identify possible breakpoints between fused genes in the processed data; filtering said possible breakpoints, to retain candidate breakpoints, wherein each candidate breakpoint is in a 5′ untranslated region (UTR) or a coding DNA sequence (CDS) of a functional gene region and each candidate breakpoint does not occur in an intron; and annotating the candidate breakpoints with at least one annotation useful in determining a relevance of a gene fusion for at least one of cancer diagnosis, cancer prognosis, or cancer treatment, wherein the gene fusion comprises the candidate breakpoint.

In one embodiment, a non-transitory computer readable program storage unit is provided, the non-transitory computer readable program storage unit encoded with instructions that, when executed by a computer, perform a method, comprising receiving mutation data from at least one cancer cell line; annotating the mutation data with at least one of variant classification, variant position, or variant change, to yield annotated mutation data; filtering the annotated mutation data, to yield gene region mutation data; classifying the gene region mutation data as hotspot, deleterious, or other; and nominating a gene comprising the gene region mutation as a gain of function, loss of function, or recurrent other gene, based on the relative frequency of mutations in the gene and the classifications of all gene region mutations in the gene.

In one embodiment, a method is provided, the method comprising detecting one or more gene fusions in a sample from a subject, to yield gene fusion detection data, wherein at least one of the gene fusions is selected from the gene fusions listed in Tables 1-3, 19, and 22, receiving by a computer system the gene fusion detection data, and identifying by the computer system at least one therapeutic option recommended for the subject, based on the gene fusion detection data.

In one embodiment, a system is provided, the system comprising a detector configured to (i) detect one or more gene fusions in a sample from a subject, to yield gene fusion detection data, wherein at least one of the gene fusions is selected from the gene fusions listed in Tables 1-3, 19, and 22 and (ii) transmit the gene fusion detection data; and a computer system configured to receive the gene fusion detection data and identify at least one therapeutic option recommended for the subject, based on the gene fusion detection data.

In another embodiment, a novel TP53 WT gene signature is provided as well as methods of detecting expression levels of one or more of the TP53 WT gene signature genes in Table 40.

DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a workflow for gene fusion RNASeq data processing.

FIG. 2 shows the classification scheme for gene variants for Gain of Function and Loss of Function genes.

FIG. 3 summarizes the data flow that integrates the various data types into a Genetic Event Database (GEDB).

FIG. 4 is a flowchart showing the roll up of genetic events

FIG. 5 is a graph showing the TP53 WT expression signature is significantly elevated in TP53 WT breast cancer compared to breast cancer samples harboring a TP53 point mutation.

FIG. 6 is a graph showing the TP53 WT expression signature is significantly elevated in TP53 WT lung cancer compared to lung cancer samples harboring a TP53 mutation.

FIG. 7 is a graph showing the TP53 WT expression signature is significantly elevated in HP53 WT ovarian cancer compared to ovarian cancer samples harboring a TP53 mutation.

FIG. 8 A-D are graphs depicting is Raw RPKM expression values (A-B) vs. z-score normalized values for PLXNB21 and COL7A1 in Ovarian Serous Carcinoma patients (C-D). The population-wide dips in PLXNB1 expression at exons 12, 17 and 23 are smoothed out in the normalized data. A sample predicted to harbor a fusion between these genes, the red diamond indicates the caller-predicted breakpoint exon.

FIGS. 9 A-B is a table of frequent TP53 mutations by amino acid position. Mutations displayed that occur with overall frequency in patients of >0.25% in the pan-cancer analysis. A recurrent splice site mutation was identified at the intron-exon junction affecting T-125

FIGS. 10 A-B is a table of Tp53 in-frame insertion and deletion mutations. The maximum detected in-frame insertion-deletions identified was 21 bp. Greater than 99% of non-transposon indels across the genome are <100 bp.

FIG. 11 shows an exemplary process for identifying potential cancer driver genes.

DETAILED DESCRIPTION

The disclosure provides novel gene fusions and variants, as well as novel associations of gene fusions and/or gene variants with certain types of cancers. Further provided are probes, reaction mixtures, assays and kits that relate to the gene fusions and/or variants disclosed herein.

Definitions

The term “marker” or “biomarker” refers to a molecule (typically protein, nucleic acid, carbohydrate, or lipid) that is expressed in the cell, expressed on the surface of a cancer cell or secreted by a cancer cell in comparison to a non-cancer cell, and which is useful for the diagnosis of cancer, for providing a prognosis, and for preferential targeting of a pharmacological agent to the cancer cell. Oftentimes, such markers are molecules that are overexpressed in a cancer cell in comparison to a non-cancer cell, for instance, 1-fold overexpression, 2-fold overexpression, 3-fold overexpression or more in comparison to a normal cell. Further, a marker can be a molecule that is inappropriately synthesized in the cancer cell, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a normal cell. Alternatively, such biomarkers are molecules that are underexpressed in a cancer cell in comparison to a non-cancer cell, for instance, 1-fold underexpression, 2-fold underexpression, 3-fold underexpression, or more. Further, a marker can be a molecule that is inappropriately synthesized in cancer, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a normal cell.

It will be understood by the skilled artisan that markers may be used in combination with other markers or tests for any of the uses, e.g., prediction, diagnosis, or prognosis of cancer, disclosed herein.

“Biological sample” includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. For example, the biological sample can include a Fresh-Frozen Paraffin-Embedded (FFPE) sample. Alternatively, a biological sample can include blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, bronchoalveolar lavage, cultured cells, e.g., primary cultures, explants, and transformed cells, stool, urine, etc. A biological sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, Mouse; rabbit; or a bird; reptile; or fish.

A “biopsy” refers to the process of removing a tissue sample for diagnostic or prognostic evaluation, and to the tissue specimen itself. Any biopsy technique known in the art can be applied to the diagnostic and prognostic methods of the present invention. The biopsy technique applied will depend on the tissue type to be evaluated (e.g., lung etc.), the size and type of the tumor, among other factors. Representative biopsy techniques include, but are not limited to, excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy, and bone marrow biopsy. An “excisional biopsy” refers to the removal of an entire tumor mass with a small margin of normal tissue surrounding it. An “incisional biopsy” refers to the removal of a wedge of tissue from within the tumor. A diagnosis or prognosis made by endoscopy or radiographic guidance can require a “core-needle biopsy”, or a “fine-needle aspiration biopsy” which generally obtains a suspension of cells from within a target tissue. Biopsy techniques are discussed, for example, in Harrison's Principles of Internal Medicine, Kasper, et al., eds., 16th ed., 2005, Chapter 70, and throughout Part V.

The terms “overexpress,” “overexpression,” or “overexpressed” interchangeably refer to a protein or nucleic acid (RNA) that is translated or transcribed at a detectably greater level, usually in a cancer cell, in comparison to a normal cell. The term includes overexpression due to transcription, post transcriptional processing, translation, post-translational processing, cellular localization (e.g., organelle, cytoplasm, nucleus, cell surface), and RNA and protein stability, as compared to a normal cell. Overexpression can be detected using conventional techniques for detecting mRNA (i.e., RT-PCR, PCR, hybridization) or proteins (i.e., ELISA, immunohistochemical techniques). Overexpression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a normal cell. In certain instances, overexpression is 1-fold, 2-fold, 3-fold, 4-fold or more higher levels of transcription or translation in comparison to a normal cell.

The terms “underexpress,” “underexpression,” or “underexpressed” or “downregulated” interchangeably refer to a protein or nucleic acid that is translated or transcribed at a detectably lower level in a cancer cell, in comparison to a normal cell. The term includes underexpression due to transcription, post transcriptional processing, translation, post-translational processing, cellular localization (e.g., organelle, cytoplasm, nucleus, cell surface), and RNA and protein stability, as compared to a control. Underexpression can be detected using conventional techniques for detecting mRNA (i.e., RT-PCR, PCR, hybridization) or proteins (i.e., ELISA, immunohistochemical techniques). Underexpression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or less in comparison to a control. In certain instances, underexpression is 1-fold, 2-fold, 3-fold, 4-fold or more lower levels of transcription or translation in comparison to a control.

The term “differentially expressed” or “differentially regulated” refers generally to a protein or nucleic acid that is overexpressed (upregulated) or underexpressed (downregulated) in one sample compared to at least one other sample, generally in a cancer patient compared to a sample of non-cancerous tissue in the context of the present invention.

The term “system” sets forth a set of components, real or abstract, comprising a whole where each component interacts with or is related to at least one other component within the whole.

The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serino (S), Threonine (T); and 8) Cysteine (C), Methionine (M). See, e.g., Creighton, Proteins (1984).

The phrase “specifically (or selectively) binds” when referring to a protein, nucleic acid, antibody, or small molecule compound refers to a binding reaction that is determinative of the presence of the protein or nucleic acid, such as the differentially expressed genes of the present invention, often in a heterogeneous population of proteins or nucleic acids and other biologics. In the case of antibodies, under designated immunoassay conditions, a specified antibody may bind to a particular protein at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with the selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

The phrase “functional effects” in the context of assays for testing compounds that modulate a marker protein includes the determination of a parameter that is indirectly or directly under the influence of a biomarker of the invention, e.g., a chemical or phenotypic. A functional effect therefore includes ligand binding activity, transcriptional activation or repression, the ability of cells to proliferate, the ability to migrate, among others. “Functional effects” include in vitro, in vivo, and ex vivo activities.

By “determining the functional effect” is meant assaying for a compound that increases or decreases a parameter that is indirectly or directly under the influence of a biomarker of the invention, e.g., measuring physical and chemical or phenotypic effects. Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index); hydrodynamic (e.g., shape), chromatographic; or solubility properties for the protein; ligand binding assays, e.g., binding to antibodies; measuring inducible markers or transcriptional activation of the marker; measuring changes in enzymatic activity; the ability to increase or decrease cellular proliferation, apoptosis, cell cycle arrest, measuring changes in cell surface markers. The functional effects can be evaluated by many means known to those skilled in the art, e.g., microscopy for quantitative or qualitative measures of alterations in morphological features, measurement of changes in RNA or protein levels for other genes expressed in placental tissue, measurement of RNA stability, identification of downstream or reporter gene expression (CAT, luciferase, β-gal, GFP and the like), e.g., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, etc.

“Inhibitors,” “activators,” and “modulators” of the markers are used to refer to activating, inhibitory, or modulating molecules identified using in vitro and in vivo assays of cancer biomarkers. Inhibitors are compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity or expression of cancer biomarkers. “Activators” are compounds that increase, open, activate, facilitate, enhance activation, sensitize, agonize, or up regulate activity of cancer biomarkers, e.g., agonists. Inhibitors, activators, or modulators also include genetically modified versions of cancer biomarkers, e.g., versions with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, antibodies, peptides, cyclic peptides, nucleic acids, antisense molecules, ribozymes, RNAi and siRNA molecules, small organic molecules and the like. Such assays for inhibitors and activators include, e.g., expressing cancer biomarkers in vitro, in cells, or cell extracts, applying putative modulator compounds, and then determining the functional effects on activity, as described above.

A “probe” or “probes” refers to a polynucleotide that is at least eight (8) nucleotides in length and which forms a hybrid structure with a target sequence, due to complementarity of at least one sequence in the probe with a sequence in the target region. The polynucleotide can be composed of DNA and/or RNA. Probes in certain embodiments, are detectably labeled, as discussed in more detail herein. Probes can vary significantly in size. Generally, probes are, for example, at least 8 to 15 nucleotides in length. Other probes are, for example, at least 20, 30 or 40 nucleotides long. Still other probes are somewhat longer, being at least, for example, 50, 60, 70, 80, 90 nucleotides long. Yet other probes are longer still, and are at least, for example, 100, 150, 200 or more nucleotides long. Probes can be of any specific length that falls within the foregoing ranges as well. Preferably, the probe does not contain a sequence complementary to the sequence(s) used to prime for a target sequence during the polymerase chain reaction.

The terms “complementary” or “complementarity” are used in reference to polynucleotides (that is, a sequence of nucleotides) related by the base-pairing rules. For example, the sequence “A-G-T,” is complementary to the sequence “T-C-A.” Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Alternatively, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

“Oligonucleotide” or “polynucleotide” refers to a polymeric form of nucleotides of any length, either deoxyribonucleotide or ribonucleotide. These terms include, but are not limited to, a single-, double- or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidine bases or other natural chemically, biochemically modified non-natural or derivatized nucleotide bases.

“Amplification detection assay” refers to a primer pair and matched probe wherein the primer pair flanks a region of a target nucleic acid, typically a target gene that defines an amplicon, and wherein the probe binds to the amplicon.

The terms “genetic variant” and “nucleotide variant” are used herein interchangeably to refer to changes or alterations to the reference human gene or cDNA sequence at a particular locus, including, but not limited to, nucleotide base deletions, insertions, inversions, and substitutions in the coding and noncoding regions. Deletions may be of a single nucleotide base, a portion or a region of the nucleotide sequence of the gene, or of the entire gene sequence. Insertions may be of one or more nucleotide bases. The “genetic variant” or “nucleotide variant” may occur in transcriptional regulatory regions, untranslated regions of mRNA, exons, introns, or exon/intron junctions. The “genetic variant” or “nucleotide variant” may or may not result in stop codons, frame shifts, deletions of amino acids, altered gene transcript splice forms or altered amino acid sequence.

The term “gene” refers to a polynucleotide (e.g., a DNA segment), that encodes a polypeptide and includes regions preceding and following the coding regions as well as intervening sequences (introns) between individual coding segments (exons). Parent genes or protein sequences are presented as Entrez Gene IDs or accession numbers. For example, the ZNF479 Entrez Gene ID is 90827. If any changes have been made to the sequence in the Gene ID in Entrez, the change is indicated after the Gene ID with a decimal and the number of the change (e.g., 90827.1). Further, for example, TPM1 has the accession number NM_004304.

The term “allele” or “gene allele” is used herein to refer generally to a naturally occurring gene having a reference sequence or a gene containing a specific nucleotide variant.

As used herein, “haplotype” is a combination of genetic (nucleotide) variants in a region of an mRNA or a genomic DNA on a chromosome found in an individual. Thus, a haplotype includes a number of genetically linked polymorphic variants which are typically inherited together as a unit.

As used herein, the term “amino acid variant” is used to refer to an amino acid change to a reference human protein sequence resulting from “genetic variant” or “nucleotide variant” to the reference human gene encoding the reference protein. The term “amino acid variant” is intended to encompass not only single amino acid substitutions, but also amino acid deletions, insertions, and other significant changes of amino acid sequence in the reference protein. Variants of the invention are described by the following nomenclature: [original amino acid residue/position/substituted amino acid residue]. For example, the substitution of leucine for arginine at position 76 is represented as R76L.

The term “genotype” as used herein means the nucleotide characters at a particular nucleotide variant marker (or locus) in either one allele or both alleles of a gene (or a particular chromosome region). With respect to a particular nucleotide position of a gene of interest, the nucleotide(s) at that locus or equivalent thereof in one or both alleles form the genotype of the gene at that locus. A genotype can be homozygous or heterozygous. Accordingly, “genotyping” means determining the genotype, that is, the nucleotide(s) at a particular gene locus. Genotyping can also be done by determining the amino acid variant at a particular position of a protein which can be used to deduce the corresponding nucleotide variant(s).

A set of probes typically refers to a set of primers, usually primer pairs, and/or detectably-labeled probes that are used to detect the target genetic variations. The primer pairs are used in an amplification reaction to define an amplicon that spans a region for a target genetic variation for each of the aforementioned genes. The set of amplicons are detected by a set of matched probes. In an exemplary embodiment, the invention is a set of TaqMan™ (Roche Molecular Systems, Pleasanton, Calif.) assays that are used to detect a set of target genetic variations used in the methods of the invention.

In one embodiment, the set of probes are a set of primers used to generate amplicons that are detected by a nucleic acid sequencing reaction, such as a next generation sequencing reaction. In these embodiments, for example, AmpliSEQ™ (Life Technologies/Ion Torrent, Carlsbad, Calif.) or TruSEQ™ (Illumina, San Diego, Calif.) technology can be employed. In other embodiments, the two or more probes are primer pairs.

A modified ribonucleotide or deoxyribonucleotide refers to a molecule that can be used in place of naturally occurring bases in nucleic acid and includes, but is not limited to, modified purines and pyrimidines, minor bases, convertible nucleosides, structural analogs of purines and pyrimidines, labeled, derivatized and modified nucleosides and nucleotides, conjugated nucleosides and nucleotides, sequence modifiers, terminus modifiers, spacer modifiers, and nucleotides with backbone modifications, including, but not limited to, ribose-modified nucleotides, phosphoramidates, phosphorothioates, phosphonamidites, methyl phosphonates, methyl phosp7horamidites, methyl phosphonamidites, 5′-β-cyanoethyl phosphoramidites, methylenephosphonates, phosphorodithioates, peptide nucleic acids, achiral and neutral internucleotidic linkages.

“Hybridize” or “hybridization” refers to the binding between nucleic acids. The conditions for hybridization can be varied according to the sequence homology of the nucleic acids to be bound. Thus, if the sequence homology between the subject nucleic acids is high, stringent conditions are used. If the sequence homology is low, mild conditions are used. When the hybridization conditions are stringent, the hybridization specificity increases, and this increase of the hybridization specificity leads to a decrease in the yield of non-specific hybridization products. However, under mild hybridization conditions, the hybridization specificity decreases, and this decrease in the hybridization specificity leads to an increase in the yield of non-specific hybridization products.

“Stringent conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (T_(m)) for the specific sequence at a defined ionic strength pH. The T_(m), is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_(m), 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.

Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e.g., and Current Protocols in Molecular Biology, ed.

Hybridization between nucleic acids can occur between a DNA molecule and a DNA molecule, hybridization between a DNA molecule and a RNA molecule, and hybridization between a RNA molecule and a RNA molecule.

A “mutein” or “variant” refers to a polynucleotide or polypeptide that differs relative to a wild-type or the most prevalent form in a population of individuals by the exchange, deletion, or insertion of one or more nucleotides or amino acids, respectively. The number of nucleotides or amino acids exchanged, deleted, or inserted can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more such as 25, 30, 35, 40, 45 or 50. The term mutein can also encompass a translocation, for example the fusion of the polypeptides encoded by the ALK and TPM1 genes (TPM1/ALK).

“Gene fusion” refers to a chimeric genomic DNA resulting from the fusion of at least a portion of a first gene to a portion of a second gene. The point of transition between the sequence from the first gene in the fusion to the sequence from the second gene in the fusion is referred to as the “breakpoint” or “fusion point.”

Transcription of the gene fusion results in a chimeric mRNA.

“Single nucleotide polymorphism” or “SNP” refers to a DNA sequence variation that occurs when a single nucleotide (A, T, G, or C) in the genome differs between members of a biological species or paired chromosomes in a human.

“Mutation” is defined herein as a specific change at a genomic location, i.e.: Chromosome, start, stop, reference base, alternate base, variant type (SNP, INS, DEL) etc.

“Annotation” is defined herein as a transcript-specific set of properties that describe the effect of the mutation, i.e.: Gene, transcript, variant classification, variant change, variant codon position, etc.

A “primer” or “primer sequence” refers to an oligonucleotide that hybridizes to a target nucleic acid sequence (for example, a DNA template to be amplified) to prime a nucleic acid synthesis reaction. The primer may be a DNA oligonucleotide, a RNA oligonucleotide, or a chimeric sequence. The primer may contain natural, synthetic, or modified nucleotides. Both the upper and lower limits of the length of the primer are empirically determined. The lower limit on primer length is the minimum length that is required to form a stable duplex upon hybridization with the target nucleic acid under nucleic acid amplification reaction conditions. Very short primers (usually less than 3-4 nucleotides long) do not form thermodynamically stable duplexes with target nucleic acids under such hybridization conditions. The upper limit is often determined by the possibility of having a duplex formation in a region other than the pre-determined nucleic acid sequence in the target nucleic acid. Generally, suitable primer lengths are in the range of about 10 to about 40 nucleotides long. In certain embodiments, for example, a primer can be 10-40, 15-30, or 10-20 nucleotides long. A primer is capable of acting as a point of initiation of synthesis on a polynucleotide sequence when placed under appropriate conditions.

The primer will be completely or substantially complementary to a region of the target polynucleotide sequence to be copied. Therefore, under conditions conducive to hybridization, the primer will anneal to the complementary region of the target sequence. Upon addition of suitable reactants, including, but not limited to, a polymerase, nucleotide triphosphates, etc., the primer is extended by the polymerizing agent to form a copy of the target sequence. The primer may be single-stranded or alternatively may be partially double-stranded.

“Detection,” “detectable” and grammatical equivalents thereof refers to ways of determining the presence and/or quantity and/or identity of a target nucleic acid sequence. In some embodiments, detection occurs amplifying the target nucleic acid sequence. In other embodiments, sequencing of the target nucleic acid can be characterized as “detecting” the target nucleic acid. A label attached to the probe can include any of a variety of different labels known in the art that can be detected by, for example, chemical or physical means. Labels that can be attached to probes may include, for example, fluorescent and luminescence materials.

“Amplifying,” “amplification,” and grammatical equivalents thereof refers to any method by which at least a part of a target nucleic acid sequence is reproduced in a template-dependent manner, including without limitation, a broad range of techniques for amplifying nucleic acid sequences, either linearly or exponentially. Exemplary means for performing an amplifying step include ligase chain reaction (LCR), ligase detection reaction (LDR), ligation followed by Q-replicase amplification, PCR, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid strand-based amplification (NASBA), two-step multiplexed amplifications, rolling circle amplification (RCA), recombinase-polymerase amplification (RPA)(TwistDx, Cambridg, UK), and self-sustained sequence replication (3SR), including multiplex versions or combinations thereof, for example but not limited to, OLA/PCR, PCR/OLA, LDR/PCR, PCR/PCR/LDR, PCR/LDR, LCR/PCR, PCR/LCR (also known as combined chain reaction-CCR), and the like. Descriptions of such techniques can be found in, among other places, Sambrook et al. Molecular Cloning, 3^(rd) Edition; Ausbel et al.; PCR Primer: A Laboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002), Msuih et al., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press, Totowa, N.J. (2002).

Analysis of nucleic acid markers can be performed using techniques known in the art including, without limitation, sequence analysis, and electrophoretic analysis. Non-limiting examples of sequence analysis include Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al., Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman et al., Methods Mol. Cell Biol., 3:39-42 (1992)), sequencing with mass spectrometry such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; Fu et al., Nat. Biotechnol., 16:381-384 (1998)), and sequencing by hybridization. Chee et al., Science, 274:610-614 (1996); Drmanac et al., Science, 260:1649-1652 (1993); Drmanac et al., Nat. Biotechnol., 16:54-58 (1998). Non-limiting examples of electrophoretic analysis include slab gel electrophoresis such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis. Additionally, next generation sequencing methods can be performed using commercially available kits and instruments from companies such as the Life Technologies/Ion Torrent PGM or Proton, the Illumina HiSEQ or MiSEQ, and the Roche/454 next generation sequencing system.

In some embodiments, the amount of probe that gives a fluorescent signal in response to an excited light typically relates to the amount of nucleic acid produced in the amplification reaction. Thus, in some embodiments, the amount of fluorescent signal is related to the amount of product created in the amplification reaction. In such embodiments, one can therefore measure the amount of amplification product by measuring the intensity of the fluorescent signal from the fluorescent indicator.

“Detectably labeled probe” or “detector probe” refers to a molecule used in an amplification reaction, typically for quantitative or real-time PCR analysis, as well as end-point analysis. Such detector probes can be used to monitor the amplification of the target nucleic acid sequence. In some embodiments, detector probes present in an amplification reaction are suitable for monitoring the amount of amplicon(s) produced as a function of time. Such detector probes include, but are not limited to, the 5′-exonuclease assay (TAQMAN® probes described herein (see also U.S. Pat. No. 5,538,848) various stem-loop molecular beacons (see for example, U.S. Pat. Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer, 1996, Nature Biotechnology 14:303-308), stemless or linear beacons (see, e.g., WO 99/21881), PNA Molecular Beacons™ (see, e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091), linear PNA beacons (see, for example, Kubista et al., 2001, SPIE 4264:53-58), non-FRET probes (see, for example, U.S. Pat. No. 6,150,097), Sunrise®/Amplifluor™ probes (U.S. Pat. No. 6,548,250), stem-loop and duplex Scorpion probes (Solinas et al., 2001, Nucleic Acids Research 29:E96 and U.S. Pat. No. 6,589,743), bulge loop probes (U.S. Pat. No. 6,590,091), pseudo knot probes (U.S. Pat. No. 6,589,250), cyclicons (U.S. Pat. No. 6,383,752), MGB Eclipse™ probe (Epoch Biosciences), hairpin probes (U.S. Pat. No. 6,596,490), peptide nucleic acid (PNA) light-up probes, self-assembled nanoparticle probes, and ferrocene-modified probes described, for example, in U.S. Pat. No. 6,485,901; Mhlanga et al., 2001, Methods 25:463-471; Whitcombe et al., 1999, Nature Biotechnology. 17:804-807; Isacsson et al., 2000, Molecular Cell Probes. 14:321-328; Svanvik et al., 2000, Anal Biochem. 281:26-35; Wolffs et al., 2001, Biotechniques 766:769-771; Tsourkas et al., 2002, Nucleic Acids Research. 30:4208-4215; Riccelli et al., 2002, Nucleic Acids Research 30:4088-4093; Zhang et al., 2002 Shanghai. 34:329-332; Maxwell et al., 2002, J. Am. Chem. Soc. 124:9606-9612; Broude et al., 2002, Trends Biotechnol. 20:249-56; Huang et al., 2002, Chem. Res. Toxicol. 15:118-126; and Yu et al., 2001, J. Am. Chem. Soc 14:11155-11161.

Detector probes can also include quenchers, including without limitation black hole quenchers (Biosearch), Iowa Black (IDT), QSY quencher (Molecular Probes), and Dabsyl and Dabcel sulfonate/carboxylate Quenchers (Epoch).

Detector probes can also include two probes, wherein for example a fluor is on one probe, and a quencher is on the other probe, wherein hybridization of the two probes together on a target quenches the signal, or wherein hybridization on the target alters the signal signature via a change in fluorescence. Detector probes can also comprise sulfonate derivatives of fluorescenin dyes with SO₃ instead of the carboxylate group, phosphoramidite forms of fluorescein, phosphoramidite forms of CY 5 (commercially available for example from Amersham). In some embodiments, interchelating labels are used such as ethidium bromide, SYBR® Green I (Molecular Probes), and PicoGreen® (Molecular Probes), thereby allowing visualization in real-time, or end point, of an amplification product in the absence of a detector probe. In some embodiments, real-time visualization can comprise both an intercalating detector probe and a sequence-based detector probe can be employed. In some embodiments, the detector probe is at least partially quenched when not hybridized to a complementary sequence in the amplification reaction, and is at least partially unquenched when hybridized to a complementary sequence in the amplification reaction. In some embodiments, the detector probes of the present teachings have a Tm of 63-69° C., though it will be appreciated that guided by the present teachings routine experimentation can result in detector probes with other Tms. In some embodiments, probes can further comprise various modifications such as a minor groove binder (see for example U.S. Pat. No. 6,486,308) to further provide desirable thermodynamic characteristics.

In some embodiments, detection can occur through any of a variety of mobility dependent analytical techniques based on differential rates of migration between different analyte species. Exemplary mobility-dependent analysis techniques include electrophoresis, chromatography, mass spectroscopy, sedimentation, for example, gradient centrifugation, field-flow fractionation, multi-stage extraction techniques, and the like. In some embodiments, mobility probes can be hybridized to amplification products, and the identity of the target nucleic acid sequence determined via a mobility dependent analysis technique of the eluted mobility probes, as described for example in Published P.C.T. Application WO04/46344 to Rosenblum et al., and WO01/92579 to Wenz et al. In some embodiments, detection can be achieved by various microarrays and related software such as the Applied Biosystems Array System with the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer and other commercially available array systems available from Affymetrix, Agilent, IIlumina, and Amersham Biosciences, among others (see also Gerry et al., J. Mol. Biol. 292:251-62, 1999; De Bellis et al., Minerva Biotec 14:247-52, 2002; and Stears et al., Nat. Med. 9:14045, including supplements, 2003). It will also be appreciated that detection can comprise reporter groups that are incorporated into the reaction products, either as part of labeled primers or due to the incorporation of labeled dNTPs during an amplification, or attached to reaction products, for example but not limited to, via hybridization tag complements comprising reporter groups or via linker arms that are integral or attached to reaction products. Detection of unlabeled reaction products, for example using mass spectrometry, is also within the scope of the current teachings.

“Aberration” Means a genomic structural variation or alteration of DNA. Examples include: over-/under-expression; copy number amplification/deletion; mutation; gene fusion; etc.

“Driver Event” means a genomic aberration, representing a Gain of Function (GoF) mutation, a fusion, or copy number peak.

“Recurrent” means occurrence of an event in 3 or more tumor samples.

“Mitelman” means a database of Chromosome Aberrations and Gene Fusions in Cancer manually curated from literature. goo.gl/PnXMT

Gene Fusions

TABLE 1 Gene Fusions Gene A Gene B Druggable Cancer Type Symbol Symbol orientation gene Bladder Urothelial Carcinoma ALK TPM1 TPM1/ALK ALK Colon Adenocarcinoma ALK PRKAR1A PRKAR1A/ALK ALK Cutaneous Melanoma ALK NCOA1 NCOA1/ALK ALK Ovarian Serous Cystadenocarcinoma CASR LPP LPP/CASR CASR Glioblastoma EGFR MDM2 MDM2/EGFR EGFR Lower Grade Glioma ELAVL3 FGFR3 FGFR3/ELAVL3 FGFR3 Acute Myeloid Leukemia B2M GNAS B2M/GNAS GNAS Clear Cell Renal Cell Carcinoma DOCK8 JAK2 DOCK8/JAK2 JAK2 Papillary Renal Cell Carcinoma HNF1B NOTCH1 HNF1B/NOTCH1 NOTCH1 Glioblastoma NFASC NTRK1 NFASC/NTRK1 NTRK1 Thyroid Gland Carcinoma NTRK1 SSBP2 SSBP2/NTRK1 NTRK1 Thyroid Gland Carcinoma NTRK1 SQSTM1 SQSTM1/NTRK1 NTRK1 Prostate Adenocarcinoma PIK3CA TBL1XR1 TBL1XR1/PIK3CA PIK3CA Thyroid Gland Carcinoma AKAP13 RET AKAP13/RET RET Thyroid Gland Carcinoma FKBP15 RET FKBP15/RET RET Thyroid Gland Carcinoma RET TBL1XR1 TBL1XR1/RET RET Glioblastoma CEP85L ROS1 CEP85L/ROS1 ROS1 Thyroid Gland Carcinoma ALK GTF2IRD1 GTF2IRD1/ALK ALK Ovarian Serous Cystadenocarcinoma BRS3 HTATSF1 HTATSF1/BRS3 BRS3 Invasive Breast Carcinoma CCDC132 CDH1 CDH1/CCDC132; CDH1 CCDC132/CDH1 Invasive Breast Carcinoma ERBB2 SLC29A3 ERBB2/SLC29A3 ERBB2 Thyroid Gland Carcinoma MET TFG MET/TFG; MET TFG/MET Ovarian Serous Cystadenocarcinoma MNDA NOTCH2 NOTCH2/MNDA NOTCH2 Thyroid Gland Carcinoma IRF2BP2 NTRK1 IRF2BP2/NTRK1 NTRK1 Ovarian Serous Cystadenocarcinoma EIF2C2 PTK2 EIF2C2/PTK2 PTK2 Invasive Breast Carcinoma HOXB3 RARA RARA/HOXB3 RARA Prostate Adenocarcinoma ETV4 STAT3 STAT3/ETV4 STAT3 Invasive Breast Carcinoma C17orf64 TOP1 TOP1/C17orf64 TOP1 Prostate Adenocarcinoma KIAA0753 TP53 TP53/KIAA0753 TP53 Glioblastoma GFAP VIM GFAP/VIM; VIM VIM/GFAP Thyroid Gland Carcinoma LTK UACA UACA/LTK LTK Papillary Renal Cell Carcinoma ALK STRN STRN/ALK ALK Thyroid Gland Carcinoma ALK STRN STRN/ALK ALK Cutaneous Melanoma BRAF CDC27 CDC27/BRAF BRAF Thyroid Gland Carcinoma BRAF MACF1 MACF1/BRAF BRAF Thyroid Gland Carcinoma BRAF MKRN1 MKRN1/BRAF BRAF Cutaneous Melanoma BRAF TAX1BP1 TAX1BP1/BRAF BRAF Prostate Adenocarcinoma BRAF JHDM1D JHDM1D/BRAF BRAF

TABLE 2 Gene Fusions Gene A Gene B Druggable Cancer Type Symbol Symbol Orientation gene Cutaneous Melanoma CLCN6 RAF1 CLCN6/RAF1 RAF1 Cutaneous Melanoma TRAK1 RAF1 TRAK/RAF1 RAF1 Colon Adenocarcinoma AKT1 PRKACA PRKACA/AKT1 AKT1 Endometrial Endometrioid AKT1 PRKACA PRKACA/AKT1 AKT1 Adenocarcinoma Colon Adenocarcinoma AKT2 PRKACA PRKACA/AKT2 AKT2 Lung Adenocarcinoma FYN MLL MLL/FYN FYN Lung Adenocarcinoma ECHD1 FYN ECHD1/FYN FYN Invasive Breast Carcinoma JAK2 TTC13 TTC13/JAK2 JAK2 Gastric Adenocarcinoma CAB39 ERBB2 CAB39/ERBB2 ERBB2 Endometrial Endometrioid BRAF EXOC4 EXOC4/BRAF BRAF Adenocarcinoma Invasive Breast Carcinoma HOOK3 IKBKB HOOK3/IKBKB IKBKB Invasive Breast Carcinoma CDK6 KRIT1 KRIT1/CDK6 CDK6 Gastric Adenocarcinoma CAPZA2 MET CAPZA2/MET MET Invasive Breast Carcinoma ACE MLLT6 MLLT6/ACE ACE Endometrial Endometrioid HLA-C MUC16 HLA-C/MUC16 MUC16 Adenocarcinoma Head and Neck LYN NTRK3 LYN/NTRK3 LYN, NTRK3 Squamous Cell Carcinoma Ovarian Serous Cystadenocarcinoma MUC16 OR7G2 MUC16/OR7G2 MUC16 Ovarian Serous Cystadenocarcinoma MDK RAB11B RAB11B/MDK MDK Squamous Cell Lung GADD45GIP1 RB1 RB1/GADD45GIP1 RB1 Carcinoma Gastric Adenocarcinoma PRKAR2A RHOA PRKAR2A/RHOA RHOA Cutaneous Melanoma MAPK1 SHANK3 SHANK3/MAPK1 MAPK1 Thyroid Gland Carcinoma RET SPECC1L SPECC1L/RET RET Ovarian Serous Cystadenocarcinoma IGFBP2 SPP1 IGFBP2/SPP1 IGFBP2, SPP1 Invasive Breast Carcinoma PAPD7 SRD5A1 PAPD7/SRD5A1; SRD5A1 SRD5A1/PAPD7 Glioblastoma RARA TAOK1 TAOK1/RARA RARA Gastric Adenocarcinoma CDK12 THRA THRA/CDK12 THRA Invasive Breast Carcinoma NARS2 TOP1 NARS2/TOP1 TOP1 Gastric Adenocarcinoma PTK2 TRAPPC9 PTK2/TRAPPC9; PTK2 TRAPPC9/PTK2 Invasive Breast Carcinoma CBL UBE4A CBL/UBE4A CBL Lower Grade Glioma GFAP VIM GFAP/VIM; VIM VIM/GFAP Invasive Breast Carcinoma ADAM9 WRN WRN/ADAM9 ADAM9 Colon and Rectal MAP2K2 YWHAE YWHAE/MAP2K2 MAP2K2 Adenocarcinoma Head and Neck Squamous ALK CLIP4 CLIP4/ALK ALK Cell Carcinoma Squamous Cell Lung ALK CLIP4 CLIP4/ALK ALK Carcinoma Thyroid Gland Carcinoma ALK MEMO1 MEMO1/ALK ALK Thyroid Gland Carcinoma BRAF SND1 BRAF/SND1; BRAF SND1/BRAF Thyroid Gland Carcinoma BRAF ZC3HAV1 ZC3HAV1/BRAF BRAF

TABLE 3 Gene Fusions Gene A Gene B Druggable Cancer type Cancer Type Symbol Symbol orientation gene precedent Thyroid Gland NOTCH1 SEC16A SEC16A- NOTCH1 breast cancer Carcinoma NOTCH1 Invasive Breast ERC1 RET ERC1-RET RET thyroid cancer Carcinoma Ovarian Serous CCDC170 ESR1 ESR1/CCDC170 ESR1 Invasive Breast Cystadenocarcinoma Carcinoma Head and Neck RPS6KB1 VMP1 RPS6KB1/VMP1; RPS6KB1 Invasive Breast Squamous Cell VMP1/RPS6KB1 Carcinoma Carcinoma Lung Adenocarcinoma RPS6KB1 VMP1 RPS6KB1/VMP1 RPS6KB1 Invasive Breast Carcinoma Squamous Cell Lung RPS6KB1 VMP1 RPS6KB1/VMP1 RPS6KB1 Invasive Breast Carcinoma Carcinoma Ovarian Serous RPS6KB1 VMP1 RPS6KB1/VMP1 RPS6KB1 Invasive Breast Cystadenocarcinoma Carcinoma Cutaneous Melanoma RPS6KB1 VMP1 RPS6KB1/VMP1 RPS6KB1 Invasive Breast Carcinoma Gastric RPS6KB1 VMP1 RPS6KB1/VMP1 RPS6KB1 Invasive Breast Adenocarcinoma Carcinoma

TABLE 4 Breakpoint Sequence for Table 1 5′ 5′ 3′ 3′ Table 4 Fusion Gene Gene 5′ 5′ Gene Gene 3′ 3′ Gene Name Chromosome Symbol Accession Gene Breakpoint Chrom Symbol Accession Breakpoint Breakpoint Sequence SEQ ID NO: TPM1/ chr15 TPM1 NM_000366 63,354,844 chr2 ALK NM_004304 29446394 TGCGGAGAGGTCAGTAACTAAATTG 1 ALK GAGAAAAGCATTGATGACTTAGAA G|TGTACCGCCGGAAGCACCAGGAG CTGCAAGCCATGCAGATGGAGCTGC AG PRKAR1/ chr17 PRKAR1A NM_002734 66,511,717 chr2 ALK NM_004304 29446263 CTGAGAGACCCATGGCATTCCTCAG 2 ALK GGAATACTTTGAGAGGTTGGAGAA G|ACCTCCTCCATCAGTGACCTGAAG GAGGTGCCGCGGAAAAACATCACC CT NCOA1/ chr2 NCOA1 NM_003743 24,991,142 chr2 ALK NM_004304 30143047 GTGCAACAGGTTCAGGTGTTTGCTG 3 ALK ACGTCCAGTGTACAGTGAATCTGGT| AGGCGGCTGTGGGGCTGCTCCAGTT CAATCTCAGCGAGCTGTTCAGTTGG LPP/ chr3 LPP NM_005578 188,202,492 chr3 CASR NM_000388 121972795 GAAACTTTCCTCCTCCACCACCTCTT 4 CASR GATGAAGAGGCTTTCAAAGTACAG| AAGGCATCACAGGAGGCCTCTGCAT GATGTGGCTTCCAAAGACTCAAGGA MDM2/ chr12 MDM2 NM_002392 69,203,072 chr7 EGFR NM_005228 55231426 GATGGTGCTGTAACCACCTCACAGA 5 EGFR TTCCAGCTTCGGAACAAGAGACCCT| GTGAGCCAAGGGAGTTTGTGGAGA ACTCTGAGTGCATACAGTGCCACCCA FGFR3/ chr4 FGFR3 NM_000142 1,808,638 chr19 ELAVL3 NM_001420 11577572 GCCCTCCCAGAGGCCCACCTTCAAG 6 ELAVL3 CAGCTGGTGGAGGACCTGGACCGTG |TCCTTGGTACAAATGGAGCCACTGA CGACAGCAAGACCAACCTCATCGTC B2M/ chr15 B2M NM_004048 45,003,811 chr20 GNAS NM_000516 57470667 TAGCTGTGCTCGCGCTACTCTCTCTT 7 GNAS TCTGGCCTGGAGGCTATCCAGCGT|G CTGGAGAATCTGGTAAAAGCACCAT TGTGAAGCAGATGAGGATCCTGCA DOCK8/ chr9 DOCK8 NM_203447 340,321 chr9 JAK2 NM_004972 5050686 GAGATTTTGGAATTTCCAACACGAG 8 JAK2 AAGTATATGTCCCTCACACTGTGTA| CAGTGGCGGCATGATTTTGTGCACG GATGGATAAAAGTACCTGTGACTCA HNF1B/ chr17 HNF1B NM_000458 36,099,431 chr9 NOTCH1 NM_017617 139396940 TGCCGCTCTGTACACCTGGTACGTC 9 NOTCH1 AGAAAGCAACGAGAGATCCTCCGA C|GTGAGACCGTGGAGCCGCCCCCG CCGGCGCAGCTGCACTTCATGTACG TG NFASC/ chr1 NFASC NM_015090 204,951,148 chr1 NTRK1 NM_002529 156844363 GGGAAGGGCCCTGAGCCAGAGTCC 10 NTRK1 GTCATCGGTTACTCCGGAGAAGATT A|CACTAACAGCACATCTGGAGACC CGGTGGAGAAGAAGGACGAAACAC CTT SSBP2/ chr5 SSBP2 NM_012446 80,742,687 chr1 NTRK1 NM_002529 156845312 TCCAGGAGGTGGAGGGCCACCAGG 11 NTRK1 AACACCCATCATGCCTAGTCCAGCA G|GCCCGGCTGTGCTGGCTCCAGAG GATGGGCTGGCCATGTCCCTGCATT TC SQSTM1/ chr5 SQSTM1 NM_003900 179,252,226 chr1 NTRK1 NM_002529 156844363 TTTCCTGAAGAACGTTGGGGAGAGT 12 NTRK1 GTGGCAGCTGCCCTTAGCCCTCTGG| ACACTAACAGCACATCTGGAGACCC GGTGGAGAAGAAGGACGAAACACCT TBL1XR1/ chr3 TBL1XR1 NM_024665 176,914,909 chr3 PIK3CA NM_006218 178916538 CATATAAAACTACTTTAAGGAATTA 13 PIK3CA GATGTATGGTTGTCCCAAAGCAGAA |ACCTGGAAACGGTGGCCTCCAACG CCGCTCCCCCCTCCCGGGAATGGAGG AKAP13/ chr15 AKAP13 NM_006738 86,286,839 chr10 RET NM_020630 43612067 CGCCATCTGCACCTTCCATAGCCAA 14 RET ATCAGGGTCATTGGACTCAGAACTT| GGTTCTTGGAAAAACTCTAGGAGAA GGCGAATTTGGAAAAGTGGTCAAGG FKBP15/ chr9 FKBP15 NM_015258 115,932,802 chr10 RET NM_020630 43612032 AATCTTACAATGGCAGGACCATTCT 15 RET GGGAACCATCATGAATACGATCAA G|GAGGATCCAAAGTGGGAATTCCC TCGGAAGAACTTGGTTCTTGGAAAA AC TBL1XR1/ chr3 TBL1XR1 NM_024665 176,765,103 chr10 RET NM_020630 43610136 GCCCTATATTTGCATTAAAATGGAA 16 RET TAAGAAAGGAAATTTCATCCTAAGT |GCTGGACTCCATGGAGAACCAGGT CTCCGTGGATGCCTTCAAGATCCTGG CEP85L/ chr6 CEP85L 387119 118,802,942 chr6 ROS1 NM_002944 117641193 TTAATATGCCAGAAAAAGAAAGAA 17 ROS1 AAGGAGTTAGTAACTACCGTTCAGA G|TACTCTTCCAACCCAAGAGGAGA TTGAAAATCTTCCTGCCTTCCCTCGGG CCDC132/ CCDC132 NM_017667 chr7 92,940,584 CDH1 NM_004360 chr16 68,857,494 GAATGCACCTATCTTAACAAATACA 30 CDH1 ACATTGAACGTCATAAGACTTGTTG| TTCTGGGGATTCTTGGAGGAATTCT TGCTTTGCTAATTCTGATTCTGCTG CDH1/ CDH1 NM_004360 chr16 68,857,529 CCDC132 NM_017667 chr7 92,952,923 AACATCAAAGGCAATTGGCTTAAGA 31 CCDC132 ATGTTCATCATCTGCATATATTTTC|T TAGCAAAGCAAGAATTCCTCCAAGA ATCCCCAGAATGGCAGGAATTTGC CDH1/ CDH1 NM_004360 chr16 68,857,529 CCDC132 NM_017667 chr7 92,952,923 GCAAATTCCTGCCATTCTGGGGATT 32 CCDC132 CTTGGAGGAATTCTTGCTTTGCTAA| GAAAATATATGCAGATGATGAACAT TCTTAAGCCAATTGCCTTTGATGTT EIF2C2/ EIF2C2 NM_012154 chr8 141,645,584 PTK2 NM_005607 chr8 141,685,598 GCTGCAGGATCTGGTTTACCCACAG 33 PTK2 GCTGATATATATGTTGGTTTCCAAT| CGGGGCCGGCTCCCGAGTACATGGT GGCGCCGCCGAGGGGCTCCGGGGCC EIF2C2/ EIF2C2 NM_012154 chr8 141,645,584 PTK2 NM_005607 chr8 141,685,598 GGCCCCGGAGCCCCTCGGCGGCGCC 34 PTK2 ACCATGTACTCGGGAGCCGGCCCCG |ATTGGAAACCAACATATATATCAG CCTGTGGGTAAACCAGATCCTGCAG EIF2C2/ EIF2C2 NM_012154 chr8 141,645,584 PTK2 NM_005607 chr8 141,712,806 CCCCGGAGCCCCTCGGCGGCGCCAC 35 PTK2 CATGTACTCGGGAGCCGGCCCCGGT |TTCTGGCTACCCTGGTTCACATGGA ATCACAGCCATGGCTGGCAGCATCT EIF2C2/ EIF2C2 NM_012154 chr8 141,645,584 PTK2 NM_005607 chr8 141,762,415 CGAAGTACAGTTTTTACATGTTTTA 36 PTK2 ATTGCAACCGCCAAAGCTGGATTCT| CCGGGGCCGGCTCCCGAGTACATGG TGGCGCCGCCGAGGGGCTCCGGGG EIF2C2/ EIF2C2 NM_012154 chr8 141,645,584 PTK2 NM_005607 chr8 141,675,096 GGCCCCGGAGCCCCTCGGCGGCGCC 37 PTK2 ACCATGTACTCGGGAGCCGGCCCCG |GAAGTCGGCTTGGCCCTGAGGACA TTATTGGCCACTGTGGATGAGACCAT ERBB2/ ERBB2 NM_004448 chr17 37,883,211 SLC29A3 NM_018344 chr10 73,115,986 ACACATGGGCCGCAAGAACAGGCC 38 SLC29A3 TCATGTAGTACCTGGCATACTCCAG C|GCCCGGGGCAGGGTCTGGACAGA AGAAGCCCTGCTGGGGTACCAGATA CT ERBB2/ ERBB2 NM_004448 chr17 37,883,548 SLC29A3 NM_018344 chr10 73,121,774 GGGCGCTGGGGGCATGGTCCACCAC 39 SLC29A3 AGGCACCGCAGCTCATCTACCAGGA |CTCCCTCAGTGCCCCTTCGGTGGCC TCCAGATTCATTGATTCCCACACAC ERBB2/ ERBB2 NM_004448 chr17 37,883,598 SLC29A3 NM_018344 chr10 73,121,726 GTGGCGGTGGGGACCTGACACTAG 40 SLC29A3 GGCTGGAGCCCTCTGAAGAGGAGG CC|TGTTCTTGCGGCCCATGTGTTTT CTGGTGAAGAGGAGCTTCCCCAGGA CT ERBB2/ ERBB2 NM_004448 chr17 37,883,205 SLC29A3 NM_018344 chr10 73,115,911 CTGAGGAGTATCTGGTACCCCAGCA 41 SLC29A3 GGGCTTCTTCTGTCCAGACCCTGCC| CAGCGCCCTGGCCTTCTTCCTGACG GCCACTGTCTTCCTCGTGCTCTGCA ERBB2/ ERBB2 NM_004448 chr17 37,882,078 SLC29A3 NM_018344 chr10 73,115,911 TGCAGAGCACGAGGAAGACAGTGG 42 SLC29A3 CCGTCAGGAAGAAGGCCAGGGCGC TG|GGTGCAGATGGGGGGCTGGGGC AGCCGCTCCCCCTTTTCCAGCAGGT CAG GFAP/ GFAP NM_002055 chr17 42,987,987 VIM NM_003380 chr10 17,277,377 AGGAGAACCGGATCACCATTCCCGT 43 VIM GCAGACCTTCTCCAACCTGCAGATT| CGAGGAGAGCAGGATTCTCTGCCTC TTCCAAACTTTTCCTCCCTGAACCT GFAP/ GFAP NM_002055 chr17 42,988,732 VIM NM_003380 chr10 17,277,285 ACGTGCGGGAGGCGGCCAGTTATCA 44 VIM GGAGGCGCTGGCGCGGCTGGAGGA A|ATGGCTCGTCACCTTCGTGAATAC CAAGACCTGCTCAATGTTAAGATGGC GFAP/ GFAP NM_002055 chr17 42,987,987 VIM NM_003380 chr10 17,277,377 AGGAGAACCGGATCACCATTCCCGT 45 VIM GCAGACCTTCTCCAACCTGCAGATT| CGAGGAGAGCAGGATTTCTCTGCCT CTTCCAAACTTTTCCTCCCTGAACC GFAP/ GFAP NM_002055 chr17 42,988,622 VIM NM_003380 chr10 17,277,371 AATGTCAAGCTGGCCCTGGACATCG 46 VIM AGATCGCCACCTACAGGAAGCTGCT |GGAAGGCGAGGAGAGCAGGATTTC TCTGCCTCTTCCAAACTTTTCCTCCC GFAP/ GFAP NM_002055 chr17 42,985,511 VIM NM_003380 chr10 17,277,237 ATCACCATTCCCGTGCAGACCTTCT 47 VIM CCAACCTGCAGATTCGAGAAACCAG |GACACTATTGGCCGCCTGCAGGAT GAGATTCAGAATATGAAGGAGGAA AT GFAP/ GFAP NM_001131019 chr17 42,987,602 VIM NM_003380 chr10 17,277,286 CTTCTCCAACCTGCAGATTCGAGGG 48 VIM GGCAAAAGCACCAAAGACGGGGAA A|TGGCTCGTCACCTTCGTGAATACC AAGACCTGCTCAATGTTAAGATGGCC GFAP/ GFAP NM_002055 chr17 42,987,983 VIM NM_003380 chr10 17,278,298 GAACCGGATCACCATTCCCGTGCAG 49 VIM ACCTTCTCCAACCTGCAGATTCGAG| AATCTGGATTCACTCCCTCTGGTTG ATACCCACTCAAAAAGGACACTTCT GFAP/ GFAP NM_002055 chr17 42,992,594 VIM NM_003380 chr10 17,271,785 CAGAGATGATGGAGCTCAATGACC 50 VIM GCTTTGCCAGCTACATCGAGAAGGT T|CGCTTCCTGGAGCAGCAGAATAA GATCCTGCTGGCCGAGCTCGAGCAG CT GFAP/ GFAP NM_002055 chr17 42,985,469 VIM NM_003380 chr10 17,277,285 GAAACCAGCCTGGACACCAAGTCTG 51 VIM TGTCAGAAGGCCACCTCAAGAGGA A|ATGGCTCGTCACCTTCGTGAATAC CAAGACCTGCTCAATGTTAAGATGGC GFAP/ GFAP NM_002055 chr17 42,988,779 VIM NM_003380 chr10 17,277,168 CACGAACGAGTCCCTGGAGAGGCA 52 VIM GATGCGCGAGCAGGAGGAGCGGCA CG|AATGAGTCCCTGGAACGCCAGA TGCGTGAAATGGAAGAGAACTTTGC CGT GFAP/ GFAP NM_002055 chr17 42,988,637 VIM NM_003380 chr10 17,277,351 GGCAGAGAAATCCTGCTCTCCTCGC 53 VIM CTTCCAGCAGCTTCCTGTAGGTGGC| GTGGCGATCTCGATGTCCAGGGCCA GCTTGACATTGAGCAGGTCCTGGTA GFAP/ GFAP NM_002055 chr17 42,992,627 VIM NM_003380 chr10 17,271,752 CTGGCTTCAAGGAGACCCGGGCCAG 54 VIM TGAGCGGGCAGAGATGATGGAGCT C|AATGACCGCTTCGCCAACTACATC GACAAGGTGCGCTTCCTGGAGCAGCA GFAP/ GFAP NM_002055 chr17 42,988,742 VIM NM_003380 chr10 17,277,351 GAGGAGCGGCACGTGCGGGAGGCG 55 VIM GCCAGTTATCAGGAGGCGCTGGCGC G|GCCACCTACAGGAAGCTGCTGGA AGGCGAGGAGAGCAGGATTTCTCTG CC GFAP/ GFAP NM_002055 chr17 42,988,025 VIM NM_003380 chr10 17,276,771 CATCGAGATCGCCACCTACAGGAAG 56 VIM CTGCTAGAGGGCGAGGAGAACCGG A|GACAGGTGCAGTCCCTCACCTGTG AAGTGGATGCCCTTAAAGGAACCA AT GFAP/ GFAP NM_002055 chr17 42,988,742 VIM NM_003380 chr10 17,277,367 GAGGAGCGGCACGTGCGGGAGGCG 57 VIM GCCAGTTATCAGGAGGCGCTGGCGC G|TGCTGGAAGGCGAGGAGAGCAGG ATTTCTCTGCCTCTTCCAAACTTTTCC GFAP/ GFAP NM_002055 chr17 42,988,642 VIM NM_003380 chr10 17,277,351 GGCAGAGAAATCCTGCTCTCCTCGC 58 VIM CTTCCAGCAGCTTCCTGTAGGTGGC| GATCTCGATGTCCAGGGCCAGCTTG ACATTGAGCAGGTCCTGGTACTCCT GFAP/ GFAP NM_002055 chr17 42,988,642 VIM NM_003380 chr10 17,277,351 AGGAGTACCAGGACCTGCTCAATGT 59 VIM CAAGCTGGCCCTGGACATCGAGATC |GCCACCTACAGGAAGCTGCTGGAA GGCGAGGAGAGCAGGATTTCTCTGC GFAP/ GFAP NM_002055 chr17 42,992,612 VIM NM_003380 chr10 17,271,824 CCCGGGCCAGTGAGCGGGCAGAGA 60 VIM TGATGGAGCTCAATGACCGCTTTGC C|CTCGAGCAGCTCAAGGGCCAAGG CAAGTCGCGCCTGGGGGACCTCTAC GA GFAP/ GFAP NM_002055 chr17 42,992,610 VIM NM_003380 chr10 17,271,769 CGGGCCAGTGAGCGGGCAGAGATG 61 VIM ATGGAGCTCAATGACCGCTTTGCCA G|CTACATCGACAAGGTGCGCTTCCT GGAGCAGCAGAATAAGATCCTGCT GG GTF2IRD1/ GTF2IRD1 NM_005685 chr7 73,935,627 ALK NM_004304 chr2 29,446,394 ACGTCCATGCCTCCAAGCGCATTCT 62 ALK CTTCTCCATCGTCCATGACAAGTCA| GTGTACCGCCGGAAGCACCAGGAG CTGCAAGCCATGCAGATGGAGCTGCA HTATSF1/ HTATSF1 NM_014500 chrX 135,586,622 BRS3 NM_001727 chrX 135,572,292 CCATGAGCGAGTTGTCATCATCAAG 63 BRS3 AATATGTTTCATCCTATGGATTTTG| AGATACAAGGCAGTTGTGAAGCCA CTTGAGCGACAGCCCTCCAATGCCAT IRF2BP2/ IRF2BP2 NM_182972 chr1 234,744,241 NTRK1 NM_002529 chr1 156,844,363 CTCGGGGCCCTTCGAGAGCAAGTTT 64 NTRK1 AAGAAGGAGCCGGCCCTGACTGCA G|ACACTAACAGCACATCTGGAGAC CCGGTGGAGAAGAAGGACGAAACA CCT IRF2BP2/ IRF2BP2 NM_182972 chr1 234,744,241 NTRK1 NM_002529 chr1 156,844,363 AGGTGTTTCGTCCTTCTTCTCCACCG 65 NTRK1 GGTCTCCAGATGTGCTGTTAGTGT|C TGCAGTCAGGGCCGGCTCCTTCTTA AACTTGCTCTCGAAGGGCCCCGAG MET/ MET NM_000245 chr7 116,412,043 TFG NM_006070 chr3 100,455,420 AGAAATGGTTTCAAATGAATCTGTA 66 TFG GACTACCGAGCTACTTTTCCAGAAG| GGCCACCCAGTGCTCCTGCAGAAGA TCGTTCAGGAACACCCGACAGCATT MET/ MET NM_000245 chr7 116,412,013 TFG NM_006070 chr3 100,455,435 TGTAAGTGCCCGAAGTGTAAGCCCA 67 TFG ACTACAGAAATGGTTTCAAATGAAT |CTGCAGAAGATCGTTCAGGAACAC CCGACAGCATTGCTTCCTCCTCCTCA MET/ MET NM_000245 chr7 116,414,937 TFG NM_006070 chr3 100,455,447 AATGGTTTCAAATGAATCTGTAGAC 68 TFG TACCGAGCTACTTTTCCAGAAGATC| GTTCAGGAACACCCGACAGCATTGC TTCCTCCTCCTCAGCAGCTCACCCA MET/ MET NM_000245 chr7 116,415,078 TFG NM_006070 chr3 100,455,435 TATATCCAGTCCATTACTGCAAAAT 69 TFG ACTGTCCACATTGACCTCAGTGCTC| CTGCAGAAGATCGTTCAGGAACACC CGACAGCATTGCTTCCTCCTCCTCA NOTCH2/ NOTCH2 NM_024408 chr1 120,478,095 MNDA NM_002432 chr1 158,815,377 TATTGACCTTGTGAACCATTTCAAG 70 MNDA TGCTCTTGCCCACCAGGCACTCGGG| AATCAGGAAACCCAGGCCCAACGG CAGGTGGATGCAAGAAGAAATGTT CC NOTCH2/ NOTCH2 NM_024408 chr1 120,478,095 MNDA NM_002432 chr1 158,815,377 GTATTGACCTTGTGAACCATTTCAA 71 MNDA GTGCTCTTGCCCACCAGGCACTCGG| GAATCAGGAAACCCAGGCCCAACG GCAGGTGGATGCAAGAAGAAATGT TC RARA/ RARA NM_000964 chr17 38,508,759 HOXB3 NM_002146 chr17 46,632,980 CCATCGCCGACCAGATCACCCTCCT 72 HOXB3 CAAGGCTGCCTGCCTGGACATCCTG| GAGGGGAGATTTGTCGCCTGCCGCT CGCTCTGGGGCTCGATGTGAATATA STAT3/ STAT3 NM_003150 chr17 40,468,807 ETV4 NM_001986 chr17 41,611,353 GTTTGGAAATAATGGTGAAGGTGCT 73 ETV4 GAACCCTCAGCAGGAGGGCAGTTTG |TAGCTTTCCACAGCCCCACCACCAG GATCAAGAAGGAGCCCCAGAGTCCC STAT3/ STAT3 NM_003150 chr17 40,468,860 ETV4 NM_001986 chr17 41,613,825 AGCAATACCATTGACCTGCCGATGT 74 ETV4 CCCCCCGCACTTTAGATTCATTGAT| GCAGTTTGTTCCTGATTTCCATTCAG AAAACCTAGCTTTCCACAGCCCCA STAT3/ STAT3 NM_003150 chr17 40,468,846 ETV4 NM_001986 chr17 41,610,042 CCTGCCGATGTCCCCCCGCACTTTA 75 ETV4 GATTCATTGATGCAGTTTGGAAATA| GATGTCACCGGGTGCGCATCAATGT ACCTCCACACAGAGGGCTTCTCTGG TFG/ TFG NM_006070 chr3 100,451,516 MET NM_000245 chr7 116,414,935 ATCAATAAAAATGTTATGTCAGCGT 76 MET TTGGCTTAACAGATGATCAGGTTTC| AGATCAGTTTCCTAATTCATCTCAG AACGGTTCATGCCGACAAGTGCAGT TOP1/ TOP1 NM_003286 chr20 39,729,993 C17orf64 NM_181707 chr17 58,503,144 CATCCAAGGTTCCATTAAATACATC 77 C17orf64 ATGCTTAACCCTAGTTCACGAATCA| AGGTGACAAATGTGTCATGCCTGGA GACAAGCTCCAGCGCCAGCCCTGCT TOP1/ TOP1 NM_003286 chr20 39,729,993 C17orf64 NM_181707 chr17 58.503.144 CCAAGGTTCCATTAAATACATCATG 78 C17orf64 CTTAACCCTAGTTCACGAATCAAGG| TGACAAATGTGTCATGCCTGGAGAC AAGCTCCAGCGCCAGCCCTGCTAGA TOP1/ TOP1 NM_003286 chr20 39,728,797 C17orf64 NM_181707 chr17 58,503,167 TGGCATGGCGCATGAGCGAGTCTCT 79 C17orf64 AGCAGGGCTGGCGCTGGAGCTTGTC |TCCAGGAGGCTCTATCTTGAAGTTA GCAATCCTCTCTTTGTGGTTATCCA TP53/ TP53 NM_000546 chr17 7,590,695 KIAA0753 NM_014804 chr17 6,498,373 TCAGCATATGCGATTTTATTATATCT 80 KIAA0753 TTGACGAACAGACTCCTGGTATTT|C CAATCCAGGGAAGCGTGTCACCGTC GTGGAAAGCACGCTCCCAGCCCGA TP53/ TP53 NM_000546 chr17 7,579,529 KIAA0753 NM_014804 chr17 6,493,323 TCCCAAGCAATGGATGATTTGATGC 81 KIAA0753 TGTCCCCGGACGATATTGAACAAT TTCCCTGGATGAAAGTGTGGGAACA GAGGAAGGATCAGAGAAAAGAGAGG TP53/ TP53 NM_000546 chr17 7,590,695 KIAA0753 NM_014804 chr17 6,498,373 TTCGGGCTGGGAGCGTGCTTTCCAC 82 KIAA0753 GACGGTGACACGCTTCCCTGGATTG| GAAATACCAGGAGTCTGTTCGTCAA AGATATAATAAAATCGCATATGCTG VIM/ VIM NM_003380 chr10 17,277,255 GFAP NM_002055 chr17 42,987,987 GAACTTTGCCGTTGAAGCTGCTAAC 83 GFAP TACCAAGACACTATTGGCCGCCTGC| TCGAGAAACCAGCCTGGACACCAA GTCTGTGTCAGAAGGCCACCTCAAGA VIM/ VIM NM_003380 chr10 17,277,325 GFAP NM_002055 chr17 42,988,666 AAGGAGGAAATGGCTCGTCACCTTC 84 GFAP GTGAATACCAAGACCTGCTCAATGT| CAAGCTGGCCCTGGACATCGAGATC GCCACCTACAGGAAGCTGCTAGAGG VIM/ VIM NM_003380 chr10 17,277,255 GFAP NM_002055 chr17 42,987,987 TTGAAGCTGCTAACTACCAAGACAC 85 GFAP TATTGGCCGCCTGC|TCGAGAAACCA GCCTGGACACCAAGTCTGTGTCAGA AGGCCACCTCAAGA VIM/ VIM NM_003380 chr10 17,277,370 GFAP NM_002055 chr17 42,988,621 AATGTTAAGATGGCCCTTGACATTG 86 GFAP AGATTGCCACCTACAGGAAGCTGCT |AGAGGGCGAGGAGAACCGGATCAC CATTCCCGTGCAGACCTTCTCCAACC VIM/ VIM NM_003380 chr10 17,271,830 GFAP NM_002055 chr17 42,992,688 GGTGCGCTTCCTGGAGCAGCAGAAT 87 GFAP AAGATCCTGCTGGCCGAGCTCGAGC |GGGCACTCAATGCTGGCTTCAAGG AGACCCGGGCCAGTGAGCGGGCAG AG VIM/ VIM NM_003380 chr10 17,277,350 GFAP NM_002055 chr17 42,988,641 GTGAATACCAAGACCTGCTCAATGT 88 GFAP TAAGATGGCCCTTGACATTGAGATT| GCCACCTACAGGAAGCTGCTAGAG GGCGAGGAGAACCGGATCACCATT CC VIM/ VIM NM_003380 chr10 17,277,877 GFAP NM_002055 chr17 42,988,655 GAAGGCGAGGAGAGCAGGATTTCT 89 GFAP CTGCCTCTTCCAAACTTTTCCTCCCT| TGGACATCGAGATCGCCACCTACAG GAAGCTGCTAGAGGGCGAGGAGAAC VIM/ VIM NM_003380 chr10 17,277,877 GFAP NM_002055 chr17 42,988,655 GGAAGGCGAGGAGAGCAGGATTCT 90 GFAP CTGCCTCTTCCAAACTTTTCCTCCCT| TGGACATCGAGATCGCCACCTACAG GAAGCTGCTAGAGGGCGAGGAGAAC UACA/ chr15 UACA NM_018003 70,957,001 chr15 LTK NM_002344 41799372 TGATTGACACTCTGCAGCACCAAGT 184 LTK GAAATCTCTGGAGCAACAGCTGGCC |GTGGGGCTTGGCCCGGCCCAGTCCT GGCCTCTGCCACCAGGTGTCACCGA STRN/ chr2 STRN NM_003162 37,143,221 chr2 ALK NM_004304 29446394 TACGGGACAGAATTGAATCAGGGA 185 ALK GATATGAAGCCTCCAAGCTATGATT C|TGTGTACCGCCGGAAGCACCAGG AGCTGCAAGCCATGCAGATGGAGCT GC STRN/ chr2 STRN NM_003162 37,143,221 chr2 ALK NM_004304 29446394 TACGGGACAGAATTGAATCAGGGA 186 ALK GATATGAAGCCTCCAAGCTATGATT C|TGTGTACCGCCGGAAGCACCAGG AGCTGCAAGCCATGCAGATGGAGCT GC JHDM1D/ chr7 JHDM1D NM_030647 139,810,895 chr7 BRAF NM_004333 140481493 TAGACCTGGACACCTTATTAAAGAA 187 BRAF CTTTCTAAAGTAATTCGAGCAATAG| AGAAAACACTTGGTAGACGGGACT CGAGTGATGATTGGGAGATTCCTGAT JHDM1D/ chr7 JHDM1D NM_030647 139,810,895 chr7 BRAF NM_004333 140481493 GACCTGGACACCTTATTAAAGAACT 188 BRAF TTCTAAAGTAATTCGAGCAATAGAG |AAAACACTTGGTAGACGGGACTCG AGTGATGATTGGGAGATTCCTGATGG TAX1BP1/ chr7 TAX1BP1 NM_006024 27,827,222 chr7 BRAF NM_004333 140481493 CTGAAAAGGAAAATCTGCAAAGAA 189 BRAF CTTTCCTGCTTACAACCTCAAGTAA A|AAAACACTTGGTAGACGGGACTC GAGTGATGATTGGGAGATTCCTGAT GG MKRN1/ chr7 MKRN1 NM_013446 140,158,807 chr7 BRAF NM_004333 140487384 TGCAGGTCCTGCATCCAATGGATGC 190 BRAF TGCCCAGAGATCGCAGCATATCAAA |GACTTGATTAGAGACCAAGGATTTC GTGGTGATGGAGGATCAACCACAGG MACF1/ chr1 MACF1 NM_012090 39,896,580 chr7 BRAF NM_004333 140487384 TTGGACAAAGGGTGGATGAAATTG 191 BRAF ATGCTGCTATTCAGAGATCACAACA G|GACTTGATTAGAGACCAAGGATT TCGTGGTGATGGAGGATCAACCACA GG CDC27/ chr17 CDC27 NM_001256 45,206,816 chr7 BRAF NM_004333 140487365 CAGAGAAGGCTTTGGATACCCTAAA 192 BRAF CAAAGCCATTGTCATTGATCCCAAG| GATTTCGTGGTGATGGAGGATCAAC CACAGGTTTGTCTGCTACCCCCCCT

TABLE 5 Breakpoint sequences for Table 2 5′ 3′ Table 5 Fusion 5′ Gene 5′ Gene 5′ Gene Gene 3′ 3′ 3′ Gene Cancer Name Chromosome Symbol Accession Breakpoint Chromosome Gene Accession Breakpoint Breakpoint Sequence Melanoma CLCN6/ chr1 CLCN6 NM_001286 11867247 chr3 RAF1 NM_002880 12641914 GAGAAACACAGGAGG RAF1 AGGAGGATGAGATTC TTCCAAGGAAAGACT ATGAG|GATGCAATTC GAAGTCACAGCGAAT CAGCCTCACCTTCAGC CCTGTCCAG SEQ ID NO: 18 Melanoma TRAK1/ chr3 TRAK1 NM_014965 42235390 chr3 RAF1 NM_002880 12641914 TCCAGCATCTGGGGG RAF1 CTGCTAAGGATGCCC AGCGGCAGCTCACAG CCGAG|GATGCAATTC GAAGTCACAGCGAAT CAGCCTCACCTTCAGC CCTGTCCAG SEQ ID NO: 19 Colon PRKACA/ chr19 PRKACA NM_002730 14208406 chr14 AKT1 NM_005163 1.05E+08 AGGGCCGCACTTGGA adenocarcinoma AKT1 CCTTGTGCGGCACCCC TGAGTACCTGGCCCCT GAG|GTGCTGGAGGAC AATGACTACGGCCGT GCAGTGGACTGGTGG GGGCTGGG SEQ ID NO: 20 Colon PRKACA/ chr19 PRKACA NM_002730 14208406 chr14 AKT1 NM_005163 1.05E+08 AGGGCCGCACTTGGA adenocarcinoma AKT1 CCTTGTGCGGCACCCC TGAGTACCTGGCCCCT GAG|GTGCTGGAGGAC AATGACTACGGCCGT GCAGTGGACTGGTGG GGGCTGGG SEQ ID NO: 21 Colon PRKACA/ chr19 PRKACA NM_002730 14208406 chr14 AKT1 NM_005163 1.05E+08 AGGGCCGCACTTGGA adenocarcinoma AKT1 CCTTGTGCGGCACCCC TGAGTACCTGGCCCCT GAG|GTGCTGGAGGAC AATGACTACGGCCGT GCAGTGGACTGGTGG GGGCTGGG SEQ ID NO: 22 Endometrial PRKACA/ chr19 PRKACA NM_002730 14208406 chr14 AKT1 NM_005163 1.05E+08 AGGGCCGCACTTGGA endometriosis AKT1 CCTTGTGCGGCACCCC TGAGTACCTGGCCCCT GAG|GTGCTGGAGGAC AATGACTACGGCCGT GCAGTGGACTGGTGG GGGCTGGG SEQ ID NO: 23 Colon PRKACA/ chr19 PRKACA NM_002730 14208406 chr19 AKT2 NM_001626 40742011 AGGGCCGCACTTGGA adenocarcinoma AKT2 CCTTGTGCGGCACCCC TGAGTACCTGGCCCCT GAG|GTGCTGGAGGAC AATGACTATGGCCGG GCCGTGGACTGGTGG GGGCTGGG SEQ ID NO: 24 Lung MLL/FYN chr11 MLL NM_005933 1.18E+08 chr6 FYN NM_002037 1.12E+08 CCAGGAAGCTCGATC adenocarcinoma AAATGCCCGCCTAAA GCAGCTCTCATTTGCA GGTG|GTACTTTGGAA AACTTGGCCGAAAAG ATGCTGAGCGACAGC TATTGTCCT SEQ ID NO: 25 Lung ECHDC1/ chr6 ECHDC1 NM_001002030 1.28E+08 chr6 FYN NM_002037 1.12E+08 CAAGGTTGGGCATTG adenocarcinoma FYN GGTGGAGGAGCAGAA TTTACTACAGCATGTG ATTT|CAGGGAAGGAG ATTGGTGGGAAGCCC GCTCCTTGACAACTGG AGAGACAG SEQ ID NO: 26 Breast TTC13/JAK2 chr1 TTC13 NM_024525 2.31E+08 chr9 JAK2 NM_004972 5055786 CTTCATATCAGAGGA carcinoma CTATGCAACAGCCCA TGAAGACTTTCAGCA GTCCT|CTGGAAATTG AACTTAGCTCATTAAG GGAAGCTTTGTCTTTC GTGTCATT SEQ ID NO: 27 Gastric CAB39/ chr2 CAB39 NM_016289 231,577,945 chr17 ERBB2 NM_004448 37,863,243 GGGGACAGCGACGAC Adenocarcinoma ERBB2 GCGGAGGCAGAGAAG GGAACGCCCGGCCCA GCCCC|TGTGCACCGG CACAGACATGAAGCT GCGGCTCCCTGCCAGT CCCGAGACC SEQ ID NO: 91 Gastric CAPZA2/ chr7 CAPZA2 NM_006136 116,502,704 chr7 MET NM_000245 116,435,709 CCAGAAGGAAGATGG Adenocarcinoma MET CGGATCTGGAGGAGC AGTTGTCTGATGAAG AGAAG|TGGTCCTTTG GCGTGCTCCTCTGGGA GCTGATGACAAGAGG AGCCCCACC SEQ ID NO: 92 Invasive CBL/UBE4A chr11 CBL NM_005188 119,158,656 chr11 UBE4A NM_004788 118,261,372 CAAAATCAAACCTTC Breast CTCATCTGCCAATGCC Carcinoma ATTTATTCTCTGGCTG CCA|GGGATGAGGAGA ATTTCTGTGCCACTGT GCCCAAGGATGGACG TTCCTAT SEQ ID NO: 93 Endometrial EXOC4/ chr7 EXOC4 NM_021807 133,164,892 chr7 BRAF NM_004333 140,434,570 TCTGCGAGAACAGAG Endometrioid BRAF AAGGGAGCTCTATAG Adenocarcinoma TCGGAGTGGAGAACT GCAAG|ATTCTCGCCT CTATTGAGCTGCTGGC CCGCTCATTGCCAAA AATTCACCG SEQ ID NO: 94 Low VM/GFAP chr10 VIM NM_003380 17,271,860 chr17 GFAP NM_002055 42,992,778 CCTGCTGGCCGAGCTC Grade GAGCAGCTCAAGGGC Glioma CAAGGCAAGTCGCGC CTGG|CTCCTGGCCGC CGTCTGGGTCCTGGCA CCCGCCTCTCCCTGGC TCGAATG SEQ ID NO: 95 Low VM/GFAP chr10 VIM NM_003380 17,276,745 chr17 GFAP NM_002055 42,988,692 CTGACCTCTCTGAGGC Grade TGCCAACCGGAACAA Glioma TGACGCCCTGCGCCA GGCA|CAGGAGTACCA GGACCTGCTCAATGTC AAGCTGGCCCTGGAC ATCGAGAT SEQ ID NO: 96 Low VM/GFAP chr10 VIM NM_003380 17,276,789 chr17 GFAP NM_002055 42,990,649 CAGGCAAAGCAGGAG Grade TCCACTGAGTACCGG Glioma AGACAGGTGCAGTCC CTCAC|GTACCGCTCC AAGTTTGCAGACCTG ACAGACGCTGCTGCC CGCAACGCGG SEQ ID NO: 97 Low VM/GFAP chr10 VIM NM_003380 17,276,817 chr17 GFAP NM_002055 42,988,824 TTTGCCGTTGAAGCTG Grade CTAACTACCAAGACA Glioma CTATTGGCCGCCTGCA GGA|GTACCAGGACCT GCTCAATGTCAAGCT GGCCCTGGACATCGA GATCGCCA SEQ ID NO: 98 Low VM/GFAP chr10 VIM NM_003380 17,276,817 chr17 GFAP NM_002055 42,988,824 CATTGAGATTGCCACC Grade TACAGGAAGCTGCTG Glioma GAAGGCGAGGAGAGC AGGA|GTACCAGGACC TGCTCAATGTCAAGCT GGCCCTGGACATCGA GATCGCCA SEQ ID NO: 99 Low VM/GFAP chr10 VIM NM_003380 17,277,255 chr17 GFAP NM_002055 42,987,988 GAACTTTGCCGTTGAA Grade GCTGCTAACTACCAA Glioma GACACTATTGGCCGC CTGC|TTCGAGAAACC AGCCTGGACACCAAG TCTGTGTCAGAAGGC CACCTCAAG SEQ ID NO: 100 Low VM/GFAP chr10 VIM NM_003380 17,277,259 chr17 GFAP NM_002055 42,988,687 TTTGCCGTTGAAGCTG Grade CTAACTACCAAGACA Glioma CTATTGGCCGCCTGCA GGA|GTACCAGGACCT GCTCAATGTCAAGCT GGCCCTGGACATCGA GATCGCCA SEQ ID NO: 101 Low VM/GFAP chr10 VIM NM_003380 17,277,2259 chr17 GFAP NM_002055 42,988,687 TTTGCCGTTGAAGCTG Grade CTAACTACCAAGACA Glioma CTATTGGCCGCCTGCA GGA|GTACCAGGACCT GCTCAATGTCAAGCT GGCCCTGGACATCGA GATCGCCA SEQ ID NO: 102 Low VM/GFAP chr10 VIM NM_003380 17,277,323 chr17 GFAP NM_002055 42,988,623 TGAAGGAGGAAATGG Grade CTCGTCACCTTCGTGA Glioma ATACCAAGACCTGCT CAAT|CTAGAGGGCGA GGAGAACCGGATCAC CATTCCCGTGCAGACC TTCTCCAA SEQ ID NO: 103 Low VM/GFAP chr10 VIM NM_003380 17,277,325 chr17 GFAP NM_002055 42,988,666 AAGGAGGAAATGGCT Grade CGTCACCTTCGTGAAT Glioma ACCAAGACCTGCTCA ATGT|CAAGCTGGCCC TGGACATCGAGATCG CCACCTACAGGAAGC TGCTAGAGG SEQ ID NO: 104 Low VM/GFAP chr10 VIM NM_003380 17,277,370 chr17 GFAP NM_002055 42,988,621 AATGTTAAGATGGCC Grade CTTGACATTGAGATTG Glioma CCACCTACAGGAAGC TGCT|AGAGGGCGAGG AGAACCGGATCACCA TTCCCGTGCAGACCTT CTCCAACC SEQ ID NO: 105 Low VM/GFAP chr10 VIM NM_003380 17,277,370 chr17 GFAP NM_002055 42,988,621 AATGTTAAGATGGCC Grade CTTGACATTGAGATTG Glioma CCACCTACAGGAAGC TGCT|AGAGGGCGAGG AGAACCGGATCACCA TTCCCGTGCAGACCTT CTCCAACC SEQ ID NO: 106 Low VM/GFAP chr10 VIM NM_003380 17,277,370 chr17 GFAP NM_002055 42,988,621 AATGTTAAGATGGCC Grade CTTGACATTGAGATTG Glioma CCACCTACAGGAAGC TGCT|AGAGGGCGAGG AGAACCGGATCACCA TTCCCGTGCAGACCTT CTCCAACC SEQ ID NO: 107 Low VM/GFAP chr10 VIM NM_003380 17,277,370 chr17 GFAP NM_002055 42,988,621 AATGTTAAGATGGCC Grade CTTGACATTGAGATTG Glioma CCACCTACAGGAAGC TGCT|AGAGGGCGAGG AGAACCGGATCACCA TTCCCGTGCAGACCTT CTCCAACC SEQ ID NO: 108 Low VM/GFAP chr10 VIM NM_003380 17,277,375 chr17 GFAP NM_002055 42,988,777 TAAGATGGCCCTTGA Grade CATTGAGATTGCCACC Glioma TACAGGAAGCTGCTG GAAG|GCGGGAGGCG GCCAGTTATCAGGAG GCGCTGGCGCGGCTG GAGGAAGAGG SEQ ID NO: 109 Low VM/GFAP chr10 VIM NM_003380 17,277,877 chr17 GFAP NM_002055 42,988,655 GAAGGCGAGGAGAGC Grade AGGATTTCTCTGCCTC Glioma TTCCAAACTTTTCCTC CCT|TGGACATCGAGA TCGCCACCTACAGGA AGCTGCTAGAGGGCG AGGAGAAC SEQ ID NO: 110 Low VM/GFAP chr10 VIM NM_003380 17,277,877 chr17 GFAP NM_002055 42,988,655 GAAGGCGAGGAGAGC Grade AGGATTTCTCTGCCTC Glioma TTCCAAACTTTTCCTC CCT|TGGACATCGAGA TCGCCACCTACAGGA AGCTGCTAGAGGGCG AGGAGAAC SEQ ID NO: 111 Low GFAP/VIM chr17 GFAP NM_002055 42,984,756 chr10 VIM NM_003380 17,278,322 CCTCAAGAGGAACAT Grade CGTGGTGAAGACCGT Glioma GGAGATGCGGGATGG AGAGG|GATACCCACT CAAAAAGGACACTTC TGATTAAGACGGTTG AAACTAGAGA SEQ ID NO: 112 Low GFAP/VIM chr17 GFAP NM_002055 42,985,436 chr10 VIM NM_003380 17,277,187 GGCCACCTCAAGAGG Grade AACATCGTGGTGAAG Glioma ACCGTGGAGATGCGG GATGG|AGATGCGTGA AATGGAAGAGAACTT TGCCGTTGAAGCTGCT AACTACCAA SEQ ID NO: 113 Low GFAP/VIM chr17 GFAP NM_002055 42,985,438 chr10 VIM NM_003380 17,277,380 AAGGCCACCTCAAGA Grade GGAACATCGTGGTGA Glioma AGACCGTGGAGATGC GGGAT|GGAGAGCAGG ATTTCTCTGCCTCTTC CAAACTTTTCCTCCCT GAACCTGA SEQ ID NO: 114 Low GFAP/VIM chr17 GFAP NM_002055 42,985,452 chr10 VIM NM_003380 17,277,278 CAAGTCTGTGTCAGA Grade AGGCCACCTCAAGAG Glioma GAACATCGTGGTGAA GACCG|GGAGGAAATG GCTCGTCACCTTCGTG AATACCAAGACCTGC TCAATGTTA SEQ ID NO: 115 Low GFAP/VIM chr17 GFAP NM_001131019 42,987,510 chr10 VIM NM_003380 17,277,303 TTATACCAATACAGG Grade CTCACCAGATTGTAA Glioma ATGGAACGCCGCCGG CTCGC|GAATACCAAG ACCTGCTCAATGTTAA GATGGCCCTTGACATT GAGATTGC SEQ ID NO: 116 Low GFAP/VIM chr17 GFAP NM_002055 42,987,987 chr10 VIM NM_003380 17,277,377 AGGAGAACCGGATCA Grade CCATTCCCGTGCAGAC Glioma CTTCTCCAACCTGCAG ATT|CGAGGAGAGCAG GATTTCTCTGCCTCTT CCAAACTTTTCCTCCC TGAACC SEQ ID NO: 117 Low GFAP/VIM chr17 GFAP NM_002055 42,987,987 chr10 VIM NM_003380 17,277,377 AGGAGAACCGGATCA Grade CCATTCCCGTGCAGAC Glioma CTTCTCCAACCTGCAG ATT|CGAGGAGAGCAG GATTTCTCTGCCTCTT CCAAACTTTTCCTCCC TGAACC SEQ ID NO: 118 Low GFAP/VIM chr17 GFAP NM_002055 42,988,642 chr10 VIM NM_003380 17,277,351 AGGAGTACCAGGACC Grade TGCTCAATGTCAAGCT Glioma GGCCCTGGACATCGA GATC|GCCACCTACAG GAAGCTGCTGGAAGG CGAGGAGAGCAGGAT TTCTCTGCC SEQ ID NO: 119 Low GFAP/VIM chr17 GFAP NM_002055 42,988,655 chr10 VIM NM_003380 17,277,336 GCCCGCCACTTGCAG Grade GAGTACCAGGACCTG Glioma CTCAATGTCAAGCTG GCCCT|CTTGACATTG AGATTGCCACCTACA GGAAGCTGCTGGAAG GCGAGGAGAG SEQ ID NO: 120 Endometrial HLA- chr6 HLA-C NM_002117 31,237,270 chr19 MUC16 NM_024690 8,959,665 GCATTTTCTTCCCACA Endometrioid C/MUC16 GGTGGAAAAGGAGGG Adenocarcinoma AGCTGCTCTCAGGCTG CGT|CCAGCAACAGTG CCCAGGCTACTACCA GTCACACCTAGACCT GGAGGATC SEQ ID NO: 121 Endometrial HLA- chr6 HLA-C NM_002117 31,237,270 chr19 MUC16 NM_024690 8,959,665 GCATTTTCTTCCCACA Endometrioid C/MUC16 GGTGGAAAAGGAGGG Adenocarcinoma AGCTGCTCTCAGGCTG CGT|CCAGCAACAGTG CCCAGGCTACTACCA GTCACACCTAGACCT GGAGGATC SEQ ID NO: 122 Endometrial HLA- chr6 HLA-C NM_002117 31,237,270 chr19 MUC16 NM_024690 8,959,665 GCATTTTCTTCCCACA Endometrioid C/MUC16 GGTGGAAAAGGAGGG Adenocarcinoma AGCTGCTCTCAGGCTG CGT|CCAGCAACAGTG CCCAGGCTACTACCA GTCACACCTAGACCT GGAGGATC SEQ ID NO: 123 Endometrial HLA- chr6 HLA-C NM_002117 31,237,270 chr19 MUC16 NM_024690 8,959,665 GCATTTTCTTCCCACA Endometrioid C/MUC16 GGTGGAAAAGGAGGG Adenocarcinoma AGCTGCTCTCAGGCTG CGT|CCAGCAACAGTG CCCAGGCTACTACCA GTCACACCTAGACCT GGAGGATC SEQ ID NO: 124 Endometrial HLA- chr6 HLA-C NM_002117 31,237,270 chr19 MUC16 NM_024690 8,959,665 GCATTTTCTTCCCACA Endometrioid C/MUC16 GGTGGAAAAGGAGGG Adenocarcinoma AGCTGCTCTCAGGCTG CGT|CCAGCAACAGTG CCCAGGCTACTACCA GTCACACCTAGACCT GGAGGATC SEQ ID NO: 125 Endometrial HLA- chr6 HLA-C NM_002117 31,237,270 chr19 MUC16 NM_024690 8,959,665 GCATTTTCTTCCCACA Endometrioid C/MUC16 GGTGGAAAAGGAGGG Adenocarcinoma AGCTGCTCTCAGGCTG CGT|CCAGCAACAGTG CCCAGGCTACTACCA GTCACACCTAGACCT GGAGGATC SEQ ID NO: 126 Endometrial HLA- chr6 HLA-C NM_002117 31,237,270 chr19 MUC16 NM_024690 8,959,665 GCATTTTCTTCCCACA Endometrioid C/MUC16 GGTGGAAAAGGAGGG Adenocarcinoma AGCTGCTCTCAGGCTG CGT|CCAGCAACAGTG CCCAGGCTACTACCA GTCACACCTAGACCT GGAGGATC SEQ ID NO: 127 Endometrial HLA- chr6 HLA-C NM_002117 31,237,270 chr19 MUC16 NM_024690 8,959,665 GCATTTTCTTCCCACA Endometrioid C/MUC16 GGTGGAAAAGGAGGG Adenocarcinoma AGCTGCTCTCAGGCTG CGT|CCAGCAACAGTG CCCAGGCTACTACCA GTCACACCTAGACCT GGAGGATC SEQ ID NO: 128 Endometrial HLA- chr6 HLA-C NM_002117 31,237,270 chr19 MUC16 NM_024690 8,959,665 GCATTTTCTTCCCACA Endometrioid C/MUC16 GGTGGAAAAGGAGGG Adenocarcinoma AGCTGCTCTCAGGCTG CGT|CCAGCAACAGTG CCCAGGCTACTACCA GTCACACCTAGACCT GGAGGATC SEQ ID NO: 129 Endometrial HLA- chr6 HLA-C NM_002117 31,237,270 chr19 MUC16 NM_024690 8,959,665 GCATTTTCTTCCCACA Endometrioid C/MUC16 GGTGGAAAAGGAGGG Adenocarcinoma AGCTGCTCTCAGGCTG CGT|CCAGCAACAGTG CCCAGGCTACTACCA GTCACACCTAGACCT GGAGGATC SEQ ID NO: 130 Invasive HOOK3/ chr8 HOOK3 NM_032410 42,798,568 chr8 IKBKB NM_001556 42,147,725 GATGCAGCAGAGCTT Breast IKBKB GGAAGGATGCTTCAG Carcinoma CTCATCTTAGGCTGTG CTGT|GAACTTGGCGC CCAATGACCTGCCCCT GCTGGCCATGGAGTA CTGCCAAG SEQ ID NO: 131 Invasive HOOK3/ chr8 HOOK3 NM_032410 42,798,588 chr8 IKBKB NM_001556 42,162,705 GGATGCTTCAGCTCAT Breast IKBKB CTTAGGCTGTGCTGTG Carcinoma AACTGTGAACAGAAG CAA|GCCTCTGCGCTT AGATACCTTCATGAA AACAGAATCATCCAT CGGGATCT SEQ ID NO: 132 Ovarian IGFBP2/ chr2 IGFBP2 NM_000597 217,528,783 chr4 SPP1 NM_000582 88,896,866 GGGAGCCCCCACCAT Serous SPP1 CCGGGGGGACCCCGA Cystadenocarcinoma GTGTCATCTCTTCTAC AATG|AGCAGCAGGAG GAGGCAGAGCACAGC ATCGTCGGGACCAGA CTCGTCTCA SEQ ID NO: 133 Ovarian IGFBP2/ chr2 IGFBP2 NM_000597 217,528,783 chr4 SPP1 NM_000582 88,896,866 TGAGACGAGTCTGGT Serous SPP1 CCCGACGATGCTGTG Cystadenocarcinoma CTCTGCCTCCTCCTGC TGCT|CATTGTAGAAG AGATGACACTCGGGG TCCCCCCGGATGGTG GGGGCTCCC SEQ ID NO: 134 Invasive KRIT1/ chr7 KRIT1 NM_004912 91,842,555 chr7 CDK6 NM_001259 92,462,486 ATATTTACAAAGGCA Breast CDK6 AGCCCCAGCAATCAT Carcinoma AAAGTCATCCCTGTGT ATGT|AGGAGGGCATG CCGCTCTCCACCATCC GCGAGGTGGCGGTGC TGAGGCAC SEQ ID NO: 135 Head LYN/NTRK3 chr8 LYN NM_002350 56,866,524 chr15 NTRK3 NM_002530 88,670,398 AGATCCCCCGGGAGT and CCATCAAGTTGGTGA Neck AAAGGCTTGGCGCTG Squamous GGCAG|TTTGGGGTAT Cell CCATAGCAGTTGGAC Carcinoma TTGCTGCTTTTGCCTG TGTCCTGTT SEQ ID NO: 136 Invasive MLLT6/ACE chr17 MLLT6 NM_005937 36,868,267 chr17 ACE NM_000789 61,573,755 CCACGCAGCAGGAGA Breast AGCACCCCACCCACC Carcinoma ACGAGAGGGGCCAGA AGAAG|GTACTTTGTC AGCTTCATCATCCAGT TCCAGTTCCACGAGG CACTGTGCC SEQ ID NO: 137 Invasive MLLT6/ chr17 MLLT6 NM_005937 36,868,267 chr17 ACE NM_000789 61,573,755 CCTGGCACAGTGCCTC Breast ACE GTGGAACTGGAACTG Carcinoma GATGATGAAGCTGAC AAAG|TACCTTCTTCTG GCCCCTCTCGTGGTGG GTGGGGTGCTTCTCCT GCTGCG SEQ ID NO: 138 Ovarian MUC16/ chr19 MUC16 NM_024690 9,024,134 chr19 OR7G2 NM_001005193 9,213,932 AGTGGATCTCAGAAC Serous OR7G2 CTCAGGGACTCCATCC Cystadenocarcinoma TCCCTCTCCAGCCCCA CAA|ATTCATCATCAA CAGCATGGAAGCGAG AAACCAAACAGCTAT TTCAAAAT SEQ ID NO: 139 Ovarian MUC16/ chr19 MUC16 NM_024690 9,045,564 chr19 OR7G2 NM_001005193 9,213,935 ATTTTGAAATAGCTGT Serous OR7G2 TTGGTTTCTCGCTTCC Cystadenocarcinoma ATGCTGTTGATGATGA AT|TTGTTCTTGAGGTC ACACTCTCAGAGGCC AAGGTGGACATCCCA GGTGTG SEQ ID NO: 140 Invasive NARS2/ chr11 NARS2 NM_024678 78,189,672 chr20 TOP1 NM_003286 39,721,138 GGAACTGTTCAAGGC Breast TOP1 TACAACAATGATGGT Carcinoma TCTCTCAAAATGTCCT GAAG|GCATCAAGTGG AAATTCCTAGAACAT AAAGGTCCAGTATTT GCCCCACCA SEQ ID NO: 141 Invasive SRD5A1/ chr5 SRD5A1 NM_001047 6,633,982 chr5 PAPD7 NM_006999 6,738,796 GCGCCCAACTGCATC Breast PAPD7 CTCCTGGCCATGTTCC Carcinoma TCGTCCACTACGGGC ATCG|GTACAGATATT TGGCAGCTTTAGTACA GGTCTTTATCTTCCAA CTAGCGA SEQ ID NO: 142 Invasive PAPD7/ chr5 PAPD7 NM_006999 6,746,451 chr5 SRD5A1 NM_001047 6,662,933 GGGAGAAATTTTAAT Breast SRD5A1 TACTTGAAAACCGGT Carcinoma ATTAGAATCAAAGAA GGAGG|CTTATTTGAA TACGTAACTGCAGCC AACTATTTTGGAGAA ATCATGGAGT SEQ ID NO: 143 Gastric PRKAR2A/ chr3 PRKAR2A NM_004157 48,845,082 chr3 RHOA NM_001664 49,405,981 GACGAGGACTTGGAA Adenocarcinoma RHOA GTTCCAGTTCCTAGCA GATTTAATAGACGAG TATC|AGGTAGAGTTG GCTTTGTGGGACACA GCTGGGCAGGAAGAT TATGATCGC SEQ ID NO: 144 Gastric TRAPPC9/ chr8 TRAPPC9 NM_031466 141,460,889 chr8 PTK2 NM_005607 141,900,868 CTCTGTGTCCCGTTTG Adenocarcinoma PTK2 AGAAAAAGGACTTTG TAGGACTGGACACAG ACAG|CAGAATATGAC AGATACCTAGCATCT AGCAAAATAATGGCA GCTGCTTAC SEQ ID NO: 145 Gastric PTK2/TRAPPC9 chr8 PTK2 NM_005607 142,011,224 chr8 TRAPPC9 NM_031466 141,034,176 CCGCCCCGTCGTCGTC Adenocarcinoma TGCCTTCGCTTCACGG CGCCGAGCCGCGGTC CGA|ACCCTGGAAGCT GTCCTGAATTTCAAAT ACTCTGGAGGCCCGG GCCACAC SEQ ID NO: 146 Ovarian RAB11B/MDK chr19 RAB11B NM_004218 8,468,319 chr11 MDK NM_002391 46,404,173 AGGAAGCATTCAAGA Serous ACATCCTCACAGAGA Cystadenocarcinoma TCTACCGCATCGTGTC ACAG|GTGATGGGGGC ACAGGCACCAAAGTC CGCCAAGGCACCCTG AAGAAGGCG SEQ ID NO: 147 Ovarian RAB11B/MDK chr19 RAB11B NM_004218 8,468,374 chr11 MDK NM_002391 46,404,248 GATCGCAGACCGCGC Serous TGCCCACGACGAGTC Cystadenocarcinoma CCCGGGGAACAACGT GGTGG|CCATCCGCGT CACCAAGCCCTGCAC CCCCAAGACCAAAGC AAAGGCCAAA SEQ ID NO: 148 Squamous RB1/GADD45GIP1 chr13 RB1 NM_000321 48,955,574 chr19 GADD45GIP1 NM_052850 13,065,313 AAAACATTTAGAACG Cell ATGTGAACATCGAAT Lung CATGGAATCCCTTGCA Carcinoma TGGC|CAAGATGCCAC AGATGATTGTGAACT GGCAGCAGCAGCAGC GGGAGAACT SEQ ID NO: 149 Cutaneous SHANK3/ chr22 SHANK3 NM_033517 51,115,121 chr22 MAPK1 NM_002745 22,153,417 TTTATGCCCAGAACCT Melanoma MAPK1 CATCGATGATAAGCA GTTTGCAAAGCTTCAC ACA|AAGATCTGTGAC TTTGGCCTGGCCCGTG TTGCAGATCCAGACC ATGATCA SEQ ID NO: 150 Thyroid SPECC1L/ chr22 SPECC1L NM_015330 24,734,416 chr10 RET NM_020630 43,610,055 TGCAGCTGCAATTCCT Gland RET CGAACGCCCCTGAGC Carcinoma CCAAGTCCTATGAAA ACCC|CTCCTCAGCTG AGATGACCTTCCGGA GGCCCGCCCAGGCCT TCCCGGTCA SEQ ID NO: 151 Glioblastoma TAOK1/ chr17 TAOK1 NM_020791 27,718,042 chr17 RARA NM_000964 38,504,568 GGGAGGGCTGGGCAC RARA TATCTCTTCAGAACTG CTGCTCTGGGTCTCAA TGG|CCTTTCGCCGAC AGGTCTGGGGCGGAG CAGGCAGGCGCAGCC CCCTGCAG SEQ ID NO: 152 Gastric THRA/CDK12 chr17 THRA NM_003250 38,245,586 chr17 CDK12 NM_015083 37,686,884 CAACCACCGCAAACA Adenocarcinoma CAACATTCCGCACTTC TGGCCCAAGCTGCTG ATGA|AGAGAAGAGGC CCCCTGAGCCCCCCG GACCTCCACCGCCGC CACCTCCAC SEQ ID NO: 153 Invasive WRN/ADAM9 chr8 WRN NM_000553 30,982,516 chr8 ADAM9 NM_003816 38,871,484 TCCTTGGGAATTATGG Breast GAACTGAAAAATGCT Carcinoma GTGATAATTGCAGGT CCAG|AGACCTTTTGC CTGAAGATTTTGTGGT TTATACTTACAACAAG GAAGGGA SEQ ID NO: 154 Colon YWHAE/ chr17 YWHAE NM_006761 1,303,359 chr19 MAP2K2 NM_030662 4,123,868 CGCTATGGATGATCG and MAP2K2 AGAGGATCTGGTGTA Rectal CCAGGCGAAGCTGGC Adenocarcinoma CGAGC|TGGCCCGGAG GAAGCCGGTGCTGCC GGCGCTCACCATCAA CCCTACCATC SEQ ID NO: 155 Thyroid ZC3HAV1/ chr7 ZC3HAV1 NM_020119 138,758,639 chr7 BRAF NM_004333 140482825 ACCAAGCCAGCCA Gland BRAF ATTCTGTCTTCACC Carcinoma ACCAAATGGATTTG GTATTGGAA|GAAT GAAAACACTTGGTA GACGGGACTCGAG TGATGATTGGGAGA TTCCT SEQ ID NO: 193 Thyroid BRAF/ chr7 SND1 NM_014390 127,361,454 chr7 BRAF NM_004333 140487384 TTCACCTGTCCAGC Gland SND1 ATCCGACCACCGAG Carcinoma GCTGGAGGGGGAG AACACCCAGIGACT TGATTAGAGACCAA GGATTTCGTGGTGA TGGAGGATCAACC ACAGG SEQ ID NO: 194 Thyroid BRAF/ chr7 BRAF NM_004333 140,487,348 chr7 SND1 NM_014390 127724776 GTCAATATTGATGA Gland SND1 CTTGATTAGAGACC Carcinoma AAGGATTTCGTGGT GATGGAGG|CACCC AGTTGGAGAAGCT GATGGAGAACATG CGCAATGACATTGC CAGTC SEQ ID NO: 195 Thyroid SND1/ chr7 SND1 NM_014390 127,361,454 chr7 BRAF NM_004333 140487384 CACCTGTCCAGCAT Gland BRAF CCGACCACCGAGG Carcinoma CTGGAGGGGGAGA ACACCCAGGA|CTT GATTAGAGACCAA GGATTTCGTGGTGA TGGAGGATCAACC ACAGGTT SEQ ID NO: 196 Thyroid MEMO1/ chr2 MEMO1 NM_015955 32,168,371 chr2 ALK NM_004304 29543748 GGCTTTCACAAGTA Gland ALK CAGTCTACAAAAA Carcinoma GACCTGCTAGAGCC ATTATTGCC|CCGGA AACTGCCTGTGGGT TTTTACTGCAACTT TGAAGATGGCTTCT GTG SEQ ID NO: 197 Head CLIP4/ chr2 CLIP4 NM_024692 29,404,563 chr2 ALK NM_004304 29462609 GAGGGGTCTCAGGT and ALK CCTGCTCACGAGCT Neck CCAATGAGATGGGT Squamous ACTGTTAG|GTTGA Cell AGATGCCCAGCAC Carcinoma AGACACGCCGTGG GACCGCATCATGGT GTTCT SEQ ID NO: 198 Squamous CLIP4/ALK chr2 CLIP4 NM_024692 29,404,561 chr2 ALK NM_004304 29462607 ACGAGGGGTCTCA Cell GGTCCTGCTCACGA Lung GCTCCAATGAGATG Carcinoma GGTACTGTT|AGGTT GAAGATGCCCAGC ACAGACACGCCGT GGGACCGCATCATG GTGTT SEQ ID NO: 199

TABLE 6 breakpoint sequences for Table 3 Table 6 3′ 3′ Fusion 5′ 5′ 5′ 5′ Gene Gene 3′ 3′ Name Gene Chrom Gene Symbol Accession Gene Breakpoint Chromosome Symbol Accession Gene Breakpoint Breakpoint Sequence SEQ ID NO: SEC16A- chr9 SEC16A NM_014866 139357445 chr9 NOTCH1 NM_017617 1.39E+08 ATTGATTTCACGAATG 28 NOTCH1 AGGCAGTGGAGCAGG TGGAAGAGGAGGAGT CTGG|CCCGCGATGCT CCCAGCCCGGTGAGA CCTGCCTGAATGGCG GGAAGTGTG ERC1-RET chr12 ERC1 NM_178039 1,250,953 chr10 RET NM_020630 43612032 GGACATGTTGGATGT 29 GAAGGAGCGGAAGGT TAATGTTCTTCAGAAG AAGG|AGGATCCAAAG TGGGAATTCCCTCGG AAGAACTTGGTTCTTG GAAAAACT ESR1/CCDC170 chr6 ESR1 NM_000125 152,332,929 chr6 CCDC170 NM_025059 151,907,024 CATGGAGCACCCAGG 156 GAAGCTACTGTTTGCT CCTAACTTGCTCTTGG ACA|GATGGTCTCCCA GCTTGAAGCCCAAAT ATCTGAGCTTGTTGAA CAGTTGG RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 57,990,165 chr17 VMP1 NM_030938 57,915,656 CAGAATGTTTTGAGCT 157 ACTTCGGGTACTTGGT AAAGGGGGCTATGGA AAG|TGCTGTCCCCGG CATAGGTCCATCTCTG CAGAAGCCATTTCAG GAGTACC VMP1/RPS6KB1 chr17 VMP1 NM_030938 57,915,758 chr17 RPS6KB1 NM_003161 57,987,923 GTTCATATGGTCCAAC 158 TCCCCCATGGTCCATG CTTTCATTTAACTGAC CC|TGTGGTGTGCCCA TTTCGCTTTTGTGGTG AAGCTTCTGCCGTTGA GCCTC RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 57,970,686 chr17 VMP1 NM_030938 57,915,656 AGACCTGGACCAGCC 159 AGAGGACGCGGGCTC TGAGGATGAGCTGGA GGAGG|GGTGCTGTCC CCGGCATAGGTCCAT CTCTGCAGAAGCCAT TTCAGGAGTA VMP1/RPS6KB1 chr17 VMP1 NM_030938 57,915,758 chr17 RPS6KB1 NM_003161 57,987,923 AAGTTCATATGGTCCA 160 ACTCCCCCATGGTCCA TGCTTTCATTTAACTG AC|CCTGTGGTGTGCC CATTTCGCTTTTGTGG TGAAGCTTCTGCCGTT GAGCC RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 57,970,606 chr17 VMP1 NM_030938 57,915,656 GGTACTCCTGAAATG 161 GCTTCTGCAGAGATG GACCTATGCCGGGGA CAGCA|CTTCCCTGTCT CGGAAGTCCGGGGCT GGGTAAAAGCCGTCC CGCCTCCTT RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 57,992,064 chr17 VMP1 NM_030938 57,915,656 GTAACAGGAGCAAAT 162 ACTGGGAAAATATTT GCCATGAAGGTGCTT AAAAA|GTGCTGTCCC CGGCATAGGTCCATC TCTGCAGAAGCCATTT CAGGAGTAC RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 58,003,943 chr17 VMP1 NM_030938 57,917,129 GCCTTTCAGACTGGTG 163 GAAAACTCTACCTCA TCCTTGAGTATCTCAG TGG|GAGAAAACTGGT TGTCCTGGATGTTTGA AAAGTTGGTCGTTGTC ATGGTG RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 57,970,686 chr17 VMP1 NM_030938 57,915,656 AGACCTGGACCAGCC 164 AGAGGACGCGGGCTC TGAGGATGAGCTGGA GGAGG|GGTGCTGTCC CCGGCATAGGTCCAT CTCTGCAGAAGCCAT TTCAGGAGTA RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 57,990,165 chr17 VMP1 NM_030938 57,915,656 CAGAATGTTTTGAGCT 165 ACTTCGGGTACTTGGT AAAGGGGGCTATGGA AAG|TGCTGTCCCCGG CATAGGTCCATCTCTG CAGAAGCCATTTCAG GAGTACC RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 58,003,943 chr17 VMP1 NM_030938 57,917,129 ATGCCTTTCAGACTGG 166 TGGAAAACTCTACCT CATCCTTGAGTATCTC AGT|GGGAGAAAACTG GTTGTCCTGGATGTTT GAAAAGTTGGTCGTT GTCATGG RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 58,009,009 chr17 VMP1 NM_030938 57,917,215 ATATTTATGGAAGAC 167 ACTGCCTGCTTTTACT TGGCAGAAATCTCCA TGGC|ACAAAGTTATG CCAAACGAATCCAGC AGCGGTTGAACTCAG AGGAGAAAA RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 58,009,061 chr17 VMP1 NM_030938 57,895,132 TGGGGCATTTACATCA 168 AAAGGGGATCATCTA CAGAGACCTGAAGCC GGAG|TGGTGCTGTCC CCGGCATAGGTCCAT CTCTGCAGAAGCCAT TTCAGGAGT RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 57,970,625 chr17 VMP1 NM_030938 57,915,703 TACCCAGCCCCGGAC 169 TTCCGAGACAGGGAA GCTGAGGACATGGCA GGAGT|ACCTGGAGGC TCAACGGCAGAAGCT TCACCACAAAAGCGA AATGGGCACA RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 57,970,643 chr17 VMP1 NM_030938 57,915,710 CCTGTGGTGTGCCCAT 170 TTCGCTTTTGTGGTGA AGCTTCTGCCGTTGAG CC|TCCAGGTCTATGT CAAACACTCCTGCCA TGTCCTCAGCTTCCCT GTCTCG RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 57,992,064 chr17 VMP1 NM_030938 57,886,157 AACAGGAGCAAATAC 171 TGGGAAAATATTTGC CATGAAGGTGCTTAA AAAGG|ACTTTGCCTC CCGGGCCAAACTGGC AGTTCAAAAACTAGT ACAGAAAGTT RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 58,007,535 chr17 VMP1 NM_030938 57,915,656 CTATTTATGCAGTTAG 172 AAAGAGAGGGAATAT TTATGGAAGACACTG CCTG|TGCTGTCCCCG GCATAGGTCCATCTCT GCAGAAGCCATTTCA GGAGTACC RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 57,970,625 chr17 VMP1 NM_030938 57,915,703 TGTGCCCATTTCGCTT 173 TTGTGGTGAAGCTTCT GCCGTTGAGCCTCCA GGT|ACTCCTGCCATG TCCTCAGCTTCCCTGT CTCGGAAGTCCGGGG CTGGGTA RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 57,990,165 chr17 VMP1 NM_030938 57,917,129 CCAGAATGTTTTGAGC 174 TACTTCGGGTACTTGG TAAAGGGGGCTATGG AAA|GGGAGAAAACT GGTTGTCCTGGATGTT TGAAAAGTTGGTCGTT GTCATGG RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 57,992,037 chr17 VMP1 NM_030938 57,851,147 ATGGAAAGGTTTTTCA 175 AGTACGAAAAGTAAC AGGAGCAAATACTGG GAAA|ATATTTCATGG CCAGAGCAGCTCGCC TCTCAGGTGCTGAAC CAGATGATG RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 57,970,686 chr17 VMP1 NM_030938 57,889,031 ACCTGGACCAGCCAG 176 AGGACGCGGGCTCTG AGGATGAGCTGGAGG AGGGG|ATTCCAAATC CTTTATTTGATCTGGC TGGAATAACGTGTGG ACACTTTCT RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 57,970,686 chr17 VMP1 NM_030938 57,886,157 ACCTGGACCAGCCAG 177 AGGACGCGGGCTCTG AGGATGAGCTGGAGG AGGGG|GACTTTGCCT CCCGGGCCAAACTGG CAGTTCAAAAACTAG TACAGAAAGT RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 57,970,686 chr17 VMP1 NM_030938 57,889,031 GAAAGTGTCCACACG 178 TTATTCCAGCCAGATC AAATAAAGGATTTGG AATC|CCCTCCTCCAG CTCATCCTCAGAGCCC GCGTCCTCTGGCTGGT CCAGGTC RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 57,970,686 chr17 VMP1 NM_030938 57,886,157 CTGGACCAGCCAGAG 179 GACGCGGGCTCTGAG GATGAGCTGGAGGAG GGGGA|CTTTGCCTCC CGGGCCAAACTGGCA GTTCAAAAACTAGTA CAGAAAGTTG RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 57,970,686 chr17 VMP1 NM_030938 57,886,157 CCTGGACCAGCCAGA 180 GGACGCGGGCTCTGA GGATGAGCTGGAGGA GGGGG|ACTTTGCCTC CCGGGCCAAACTGGC AGTTCAAAAACTAGT ACAGAAAGTT RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 57,970,686 chr17 VMP1 NM_030938 57,886,157 ACCTGGACCAGCCAG 181 AGGACGCGGGCTCTG AGGATGAGCTGGAGG AGGGG|GACTTTGCCT CCCGGGCCAAACTGG CAGTTCAAAAACTAG TACAGAAAGT RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 57,970,686 chr17 VMP1 NM_030938 57,886,157 CGCGGGCTCTGAGGA 182 TGAGCTGGAGGAGGG GGA|CTTTGCCTCCCG GGCCAAACTGGCAGT TCAAAAACTAGTACA GAAAGTTG RPS6KB1/VMP1 chr17 RPS6KB1 NM_003161 57,992,064 chr17 VMP1 NM_030938 57,915,656 AGTAACAGGAGCAAA 183 TACTGGGAAAATATTT GCCATGAAGGTGCTT AAAA|AGTGCTGTCCC CGGCATAGGTCCATC TCTGCAGAAGCCATTT CAGGAGTA

The disclosure provides novel gene fusions and gene fusion variants (ie, varying breakpoint locations on one or both of the partner genes) selected from those shown in Table 1-Table 3, Table 19, and Table 22 of gene fusions such as TPM1/ALK, PRKAR1A/ALK, NCOA1/ALK, LPP/CASR, MDM2/EGFR, FGFR3/ELAVL3, B2M/GNAS, DOCK8/JAK2, HNF1B/NOTCH1, NFASC/NTRK1, SSBP2/NTRK1, SQSTM1/NTRK1, TBL1XR1/PIK3CA, AKAP13/RET, FKBP15/RET, TBL1XR1/RET, CEP85L/ROS1, CLCN6/RAF1, TRAK1/RAF1, PRKACA/AKT1, PRKACA/AKT2, MLL/FYN, ECHD1/FYN, TTC13/JAK2, SEC16A/NOTCH1, ERC1/RET, GTF2IRD1/ALK, HTATSF1/BRS3, CDH1/CCDC132, CCDC132/CDH1, ERBB2/SLC29A3, MET/TFG; TFG/MET, NOTCH2/MNDA, IRF2BP2/NTRK1, EIF2C2/PTK2, RARA/HOXB3, STAT3/ETV4,and GFAP/VIM; VIM/GFAP, TOP1/C17orf64, and TP53/KIAA0753. As a result of these discoveries, the disclosure provides isolated gene fusion nucleic acids and sequences complementary thereto, amplicons, transcripts, reaction mixtures, as well as probes that specifically recognize the nucleic acid sequences of the gene fusions, sequences complementary thereto, amplicons, and transcripts. The disclosure further contemplates antisense nucleotides for use in the treatment of the associated disease.

Table 1-Table 3, Table 19, and Table 22 provide a list of the gene fusions (Gene A/Gene B) indicating the genes involved (Gene A and Gene B), the chromosome locations, the breakpoint locations, the fusion types and the distance. The gene fusions are shown with the associated TOGA disease (The Cancer Genome Atlas). The cancers are shown with 3-4 letter abbreviations which are explained in more detail in the diagnostics section.

Generally, Tables 1-3, 19, and 22 provide one or more novel gene fusions and/or associations of gene fusions with TOGA diseases. For example, Table 19 presents novel gene fusions, and Table 22 presents novel associations of gene fusions with TOGA diseases.

Tables 4-6, 20, and 23 provide the breakpoint sequences for the gene fusions in Tables 1-3, 19, and 22. The breakpoint sequences are identified as SEQ ID NO:1-289.

Assays and Kits

In certain embodiments, assays and methods of detection are provided. Methods for detecting gene fusions provided herein are known in the art. As non-limiting examples, such assays can include 5′ nuclease PCR assays (Applied Biosystems, Foster City, Calif.), next generation sequencing assays (Ion Torrent, Carlsbad Calif.; Illumina, San Diego, Calif.), or microarray assays (Skotheim et al., Molecular Cancer 2009, 8:5). In at least one embodiment, the assays or methods include at least one primer or probe that is complementary to or encodes a gene fusion and/or breakpoint in Tables 1-6.

In at least one embodiment, assays and methods of quantitating the amount of expression of a gene fusion are provided. The methods may involve quantitating expression of one or more exons. For example, TaqMan™ Gene Expression Assays can be designed for a set of known fusion transcripts for quantitative analysis. Such assays can be designed such that the primers and probe span the breakpoint region, although in certain illustrative embodiments the primers and probe are not placed directly on the breakpoint.

In certain embodiments, the disclosure provides a primer, a probe or a set of probes or primers that specifically recognize one or more of the gene fusions and/or breakpoints disclosed herein.

In one embodiment, the disclosure provides a composition and a kit comprising a set of probes that specifically recognize a gene fusion selected from Tables 1-3, 19, and 22 and/or a breakpoint in Tables 4-6, 20, and 23. The set of probes can be, for example a set of amplification primers. In another embodiment, provided herein is a composition that includes a set of primers that flank a gene fusion selected from Tables 1-3, 19, and 22 in a target nucleic acid. The reaction mixture of this embodiment can further include a detector probe that binds to either side of a breakpoint in a gene fusion selected from Tables 1-3, 19, and 22, or that binds a binding region that spans the breakpoint in a gene fusion selected from Tables 1-3, 19, and 22. The reaction mixture that includes a detector probe or does not include a detector probe, can further include a polymerase, dNTPs, and/or a uracil DNA deglycosylase (UDG). The polymerase and UDG are typically not from a human origin. The reaction mixture can further include a target nucleic acid, for example a human target nucleic acid. The human target nucleic acid can be, for example, isolated from a biological sample from a person suspected of having a cancer.

In another embodiment, provided herein is a qPCR assay, such as a TaqMan™ assay or a Molecular Beacons™ assay, that specifically amplifies and detects a target nucleic acid that includes SEQ ID NOs: 1-289.

The disclosure also provides an isolated nucleic acid comprising at least one sequence selected from SEQ ID NOs: 1-289. The isolated nucleic acid can include a first primer on a 5′ end. Furthermore, the nucleic acid can be single stranded or double stranded.

The disclosure, in other embodiments, provides a kit that includes a detector probe and/or a set of probes, for example, a set of amplification primers that specifically recognize a nucleic acid comprising a breakpoint for a gene fusion selected from Tables 1-3, 19, and 22. For example, in certain embodiments the detector probe or set of amplification primers are designed to amplify and/or detect a nucleic acid that includes at least one of SEQ ID NOs:1-289. The kit can further include, in a separate or in the same vessel, a component from an amplification reaction mixture, such as a polymerase, typically not from human origin, dNTPs, and/or UDG. Furthermore, the kit can include a control nucleic acid. For example the control nucleic acid can include a sequence that includes the break point in a gene fusion selected from Tables 1-3, 19, and 22.

In some embodiments there is provided a kit encompassing at least 2 primer pairs and 2 detectably labeled probes. In these non-limiting embodiments, the 2 primer pairs and/or 2 detectably labeled probes form 2 amplification detection assays.

The kits of the present invention may also comprise instructions for performing one or more methods described herein and/or a description of one or more compositions or reagents described herein. Instructions and/or descriptions may be in printed form and may be included in a kit insert. A kit also may include a written description of an Internet location that provides such instructions or descriptions.

In some embodiments, the kits and assays comprise one or more probes that specifically recognize a target, such as a gene fusion nucleic acid sequence. In at least one embodiment, the kits and assays are diagnostic kits and assays.

A kit comprising a set of probes, for example, a set of amplification primers that specifically recognize a nucleic acid comprising a break point from Tables 4-6, 20, and 23 is provided. The kit can further include, in a separate or in the same vessel, a component from an amplification reaction mixture, such as a polymerase, typically not from human origin, dNTPs, and/or UDG. Furthermore, the kit can include a control nucleic acid. For example the control nucleic acid can include a sequence that includes the break point selected from Tables 4-6, 20, and 23.

In another embodiment, a gene fusion is provided comprising at least one of the break points in Tables 4-6, 20, and 23.

In some embodiments, a reaction mixture and a kit are provided. In some embodiments, the kit encompasses a detectable probe that selectively binds a gene fusion. In some embodiments, the gene fusion is any one of the gene fusions in Table 4, Table 5, Table 6, Table 20, or Table 23.

Thus, in some embodiments are provided a kit encompassing a reaction mixture and a detectable probe that selectively binds a gene fusion, the gene fusion being any one of the gene fusions in Table 4, Table 5, Table 6, Table 20, or Table 23.

Diagnostics

Methods of diagnosing, treating, and detecting gene fusions and associated disease are contemplated herein. The methods can include detecting gene fusions in a subject sample.

A subject sample can be any bodily tissue or fluid that includes nucleic acids from the subject. In certain embodiments, the sample will be a blood sample comprising circulating tumor cells or cell free DNA. In other embodiments, the sample can be a tissue, such as a cancerous tissue. The cancerous tissue can be from a tumor tissue and may be fresh frozen or formalin-fixed, paraffin-embedded (FFPE).

The disease can be a cancer or tumor. Cancers can include, but are not limited to, melanoma, cervical cancer, pancreatic cancer, head and neck squamous cancer, lung adenocarcinoma, colon adenocarcinoma, uterine carcinoma, ovarian cancer, glioblastoma, low grade glioma, lung adenocarcinoma, thyroid cancer, and gastric cancer.

Cancers can include but are not limited to, bladder carcinoma, breast carcinoma, cervical cell carcinoma, colon adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, clear cell renal cell carcinoma, kidney renal papillary cell carcinoma, acute myeloid leukemia, brain lower grade glioma, liver hepatocellular carcinoma, lung adenocarcinoma, squamous cell lung carcinoma, ovarian serous adenocarcinoma, prostate adenocarcinoma, rectal adenocarcinoma, cutaneous melanoma, stomach adenocarcinoma, thyroid carcinoma, and uterine corpus endometrioid carcinoma. As used herein, BLCA=bladder carcinoma, BRCA=breast carcinoma, CESC=cervical cell carcinoma, COAD=colon adenocarcinoma, GBM=glioblastoma multiforme, HNSC=head and neck squamous cell carcinoma, KIRK=clear cell renal cell carcinoma, KIRP=kidney renal papillary cell carcinoma, LAML=acute myeloid leukemia, LGG=brain lower grade glioma, LIHC=liver hepatocellular carcinoma, LUAD=lung adenocarcinoma, LUSC=squamous cell lung carcinoma, OV=ovarian serous adenocarcinoma, PRAD=prostate adenocarcinoma, READ=rectal adenocarcinoma, SKCM=cutaneous melanoma, STAD=stomach adenocarcinoma, THCA=thyroid carcinoma, and UCEC=uterine corpus endometrioid carcinoma.

In some embodiments, a method of detecting novel gene variants or gene fusions is provided, the method encompassing a reaction mixture, wherein the novel gene variant or gene fusion is detected by the generation of an extension product.

In another embodiment, the disclosure provides diagnostics and treatment targets utilizing the disclosed gene fusions and gene variants. The gene fusions, gene variants and associated disease states provide targets for both diagnosis and treatment. For instance, the presence, absence, or increased or decreased expression of a gene fusion target or a gene variant can be used to diagnose a disease state or may be used to prognose or detect a disease state. In at least one embodiment, the gene fusion or gene variant can have a high prevalence (frequency) in a particular cancer, a medium prevalence or a low prevalence. In at least one embodiment, the gene fusion or gene variant can have a high frequency in one cancer or tumor and a low or medium prevalence in another. In at least one embodiment, the gene fusion or gene variant can have a medium or low frequency association with a cancer or tumor. In at least one embodiment, a low or medium frequency gene fusion or gene variant can be used in combination with one or more different high frequency biomarkers of cancers to help to diagnose, prognose or identify a predisposition for a disease. The methods can be used for screening for cancer in a patient or predicting the relative prospects of a particular outcome of a cancer. For example, the presence of BRCA1 or BRCA2 mutations can be analyzed in combination with the gene fusion JAK2/TTC13 for breast cancer.

A method of detecting a cancer is provided comprising amplifying a nucleic acid that spans a breakpoint in a gene fusion selected from Tables 1-3, 19, and 22, for example the nucleic acid can include a sequence selected from SEQ ID NOs: 1-289, and detecting the presence of the nucleic acid, wherein the presence of the nucleic acid indicates a cancer is present in the sample. In another method, provided herein is a method of detecting a cancer that includes generating an amplicon that includes a sequence selected from SEQ ID NOs: 1-289, and detecting the presence of the nucleic acid, wherein the presence of the nucleic acid indicates the cancer or cancer cell is present in the sample. The amplicon typically includes primers that are extended to form the amplicon. The cancer is selected from bladder carcinoma, breast carcinoma, cervical cell carcinoma, colon adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, clear cell renal cell carcinoma, kidney renal papillary cell carcinoma, acute myeloid leukemia, brain lower grade glioma, liver hepatocellular carcinoma, lung adenocarcinoma, squamous cell lung carcinoma, ovarian serous adenocarcinoma, prostate adenocarcinoma, rectal adenocarcinoma, cutaneous melanoma, stomach adenocarcinoma, thyroid carcinoma, and uterine corpus endometrioid carcinoma.

In another embodiment is a method to detect a cancer selected from bladder carcinoma, breast carcinoma, cervical cell carcinoma, colon adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, clear cell renal cell carcinoma, kidney renal papillary cell carcinoma, acute myeloid leukemia, brain lower grade glioma, liver hepatocellular carcinoma, lung adenocarcinoma, squamous cell lung carcinoma, ovarian serous adenocarcinoma, prostate adenocarcinoma, rectal adenocarcinoma, cutaneous melanoma, stomach adenocarcinoma, thyroid carcinoma, and uterine corpus endometrioid carcinoma in a sample by detecting the presence of a gene fusion selected from Tables 1-3, 19, and 22.

New Gene Fusions

Although some of the gene fusions have been previously reported, provided herein, are numerous variations of the gene fusions in which the break points differ and/or that were not previously known. Nonlimiting examples of gene fusions in which the break points differ and/or were not previously known include:TPM1/ALK, PRKAR1A/ALK, NCOA1/ALK, LPP/CASR, MDM2/EGFR, FGFR3/ELAVL3, B2M/GNAS, DOCK8/JAK2, HNF1B/NOTCH1, NFASC/NTRK1, SSBP2/NTRK1, SQSTM1/NTRK1, TBL1XR1/PIK3CA, AKAP13/RET, FKBP15/RET, TBL1XR1/RET, CEP85L/ROS1, CLCN6/RAF1, TRAK1/RAF1, PRKACA/AKT1, PRKACA/AKT2, MLL/FYN, ECHD1/FYN and TTC13/JAK2 are novel variants with the breakpoints provided in Tables 4 and 5 as SEQ ID NOs: 1-289.

Also provided herein are numerous gene fusion variants that are associated with one or more cancers.

Cancer Associations

New gene fusion associations with cancer(s) are presented herein. Some of the gene fusions may have been associated with specific cancers or disease states previously. The methods herein have identified new associations that can be used to help diagnose and/or treat the specific cancers. The gene fusions shown in Tables 1-3, 19, and 22 provide the genes involved in the fusion and the association of that gene fusion with one or more specific cancers. For example, the fusion PRKACA/AKT1 is shown to be associated with colon adenocarcinoma and endometrial endometrioid adenocarcinoma.

The gene fusions shown in Table 3 are previously known gene fusions that have been shown to be associated with new cancers. For example, SEC16A/NOTCH1 was previously identified as associated with breast cancer. Current methods identified an association of the gene fusion SEC16A/NOTCH1 with thyroid gland carcinoma. Further, ERC1/RET was previously identified as associated with thyroid cancer. Current methods identified an association of the gene fusion ERC1/RET with invasive breast carcinoma (see Tables 3 and 6).

Reaction Mixtures and Amplicons

In another embodiment, the disclosure provides a reaction mixture comprising a probe or a set of probes that specifically recognize a gene fusion selected from Table 1-Table 3, Table 19, and Table 22. The set of probes can be, for example a set of amplification primers or a labeled probe. In another embodiment, provided herein is a reaction mixture that includes a set of primers that flank a gene fusion selected from Table 1-Table 3, Table 19, and Table 22 in a target nucleic acid. For example, the set of primers can each bind to a target sequence in the human genome within 1000, 750, 500, 250, 100, 90, 80, 75, 70, 65, 50, or 25 nucleotides of opposite sides of the one of the fusion breakpoints identified in Tables 4-6, 20, and 23. The reaction mixture of this embodiment can further include a detector probe that binds to either side of a breakpoint in a gene fusion selected from Table 1-Table 3, Table 19, and Table 22, or that binds a binding region that spans the breakpoint in a gene fusion selected from Table 1-Table 3, Table 19, and Table 22, including specific embodiments where the breakpoint is identified in Tables 4-6, 20, and 23. In exemplary embodiments, the detector probe binds to a target sequence in the human genome within 1000, 750, 500, 250, 100, 90, 80, 75, 70, 60, 50, or 25 nucleotides of one of the fusion breakpoints identified in Tables 4-6, 20, and 23. The reaction mixture that includes a detector probe or does not include a detector probe, can further include a polymerase, a reverse transcriptase, dNTPs, and/or a uracil DNA deglycosylase (UDG). The polymerase, the reverse transcriptase, and the UDG are typically not from human origin. The polymerase in illustrative embodiments is a thermostable polymerase such as a Taq polymerase. In certain embodiments, the dNTPs in the reaction mixture include dUTP, and the reaction mixture can in certain examples, be devoid of dTTP.

The reaction mixture can further include a target nucleic acid, for example a human target nucleic acid. The human target nucleic acid can be, for example, isolated from a biological sample, such as a tumor sample, from a person suspected of having a cancer selected from: BLCA=bladder carcinoma, BRCA=breast carcinoma, CESC=cervical cell carcinoma, COAD=colon adenocarcinoma, GBM=glioblastoma multiforme, HNSC=head and neck squamous cell carcinoma, KIRK=clear cell renal cell carcinoma, KIRP=kidney renal papillary cell carcinoma, LAML=acute myeloid leukemia, LGG=brain lower grade glioma, LIHC=liver hepatocellular carcinoma, LUAD=lung adenocarcinoma, LUSC=squamous cell lung carcinoma, OV=ovarian serous adenocarcinoma, PRAD=prostate adenocarcinoma, READ=rectal adenocarcinoma, SKCM=cutaneous melanoma, STAD=stomach adenocarcinoma, THCA=thyroid carcinoma, and UCEC=uterine corpus endometrioid carcinoma. In certain embodiments, the target nucleic acid is from a tumor, for example a tumor of one of the cancer types listed in the preceding sentence. Furthermore, the target nucleic acid can be extracted from a biological sample from a tumor such as, for example, an FFPE sample.

The reaction mixtures of the present invention can include an amplicon. The amplicon can be for example, an isolated nucleic acid. The amplicon can be between 25 and 2500, between 25 and 2000, between 25 and 1000, between 50 and 1000, between 50 and 500, between 50 and 250, between 50 and 200, between 50 and 150, between 50 and 100, or between 50 and 75 nucleotides in length, for example.

The amplicon can have a nucleotide sequence that is identical or complementary to the target sequence in the human genome within 1000, 750, 500, 250, 100, 90, 80, 75, 70, 65, 50, or 25 nucleotides of opposite sides of the one of the fusion breakpoints identified in Tables 4-6, 20, and 23. In certain embodiments, the amplicon includes 25 to 250, 25 to 100, 25 to 75, 50 to 250, 50 to 200, 50 to 150, 50 to 100, or 50 to 75 of the nucleotide sequence provided in FIGS. 4-6 , or a complement thereof. In certain embodiments the amplicons includes sequence variants that occur in nature. For example, the amplicons may include variable nucleotide sequences that correspond to single nucleotide variants or naturally occurring alleles.

Amplicons of the present invention, in certain illustrative embodiments, have a chemical structure that is not found in nature, and/or not found in a mammal, such as a human. For example, certain illustrative amplicons include a base that is not found in nature or not found in a mammal or that may not be found bound to the type of sugar-phosphate backbone of the amplicon. For example, the amplicon might be a DNA amplicon that includes a uracil base bound to the sugar phosphate backbone, thus having a uridine residue at least at one position and in illustrative examples, at all positions that contain a thymidine residue in a template.

Accordingly, the amplicon in illustrative embodiments is a DNA amplicon that includes one or more deoxyuridine (“dU”) residues. The dU residue can be added by including such residues in the primers used to generate the amplicon. In certain embodiments the reaction mixture includes a DNA amplicon that includes one or more dU residues for every deoxythymidine residue in the corresponding human genomic sequence. These amplicons can be generated, for example, by using a dNTP mix that includes dUTP instead of dTTP when generating the amplicon using an amplification reaction such as PCR.

In certain embodiments, the amplicon includes a segment for which a corresponding sequence is not found in the human genome, such as, for example, an oligonucleotide sequence, for example a DNA barcode sequence. The non-human segment can be for example, 5-10,000, 5-5000, 5-1000, 5-500, 5-100, 5-50, 5-25, 5-10, 10-10,000, 10-5000, 10-1000, 10-500, 10-100, 10-50, or 10-25 nucleotides in length.

In certain embodiments, the amplicon includes segment that corresponds to the region of the human genome that spans an intron, but the amplicon does not include a segment corresponding to the intron.

Gene Variants (Table 7 and/or Table 11)

TABLE 11 Gain of Function mutations Pan- Disease Tumor Tumor CBI CBI Gene Start Variant Reference Seq Seq Annotation Variant Variant Variant Variant Cancer Type Symbol Chromosome Position Type Allele Allele 1 Allele 2 Source Transcript Change Position Classification Category Prostate ACOT7 1 6387379 SNP A G G Oncomine NM_007274 p.V202A p.V202 Missense_ Hotspot Adenocarcinoma Mutation Squamous ACOT7 1 6387379 SNP A G G Oncomine NM_007274 p.V202A p.V202 Missense_ Hotspot Cell Lung Mutation Carcinoma Clear Cell ACOT7 1 6387379 SNP A G G Oncomine NM_007274 p.V202A p.V202 Missense_ Hotspot Renal Cell Mutation Carcinoma Prostate ANAPC1 2 112625621 SNP G C C Oncomine NM_022662 p.P222A p.P222 Missense_ Hotspot Adenocarcinoma Mutation Medulloblastoma ANAPC1 2 112625621 SNP G C C Oncomine NM_022662 p.P222A p.P222 Missene_ Hotspot Mutation Gastric ANAPC1 2 112625621 SNP G C C Oncomine NM_022662 p.P222A p.P222 Missense_ Hotspot Adenocarcinoma Mutation Lung ANAPC1 2 112625621 SNP G C C Oncomine NM_022662 p.P222A p.P222 Missense_ Hotspot Adenocarcinoma Mutation Papillary ANAPC1 2 112625621 SNP G C C Oncomine NM_022662 p.P222A p.P222 Missense_ Hotspot Renal Cell Mutation Carcinoma Colorectal C2orf69 2 200498052 SNP G A A Oncomine NM_153689 p.R119H p.R119 Missense_ Hotspot Adenocarcinoma Mutation Gastric C2orf69 2 200789806 SNP C T T Oncomine NM_153689 p.R119C p.R119 Missense_ Hotspot Adenocarcinoma Mutation Gastric C2orf69 2 200789807 SNP G A A Oncomine NM_153689 p.R119H p.R119 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous C4orf22 4 81791162 SNP C T T Oncomine NM_152770 p.R117C p.R117 Missense_ Hotspot Melanoma Mutation Cutaneous C4orf22 4 81791162 SNP C T T Oncomine NM_152770 p.R117C p.R117 Missense_ Hotspot Melanoma Mutation Cutaneous C4orf22 4 81504291 SNP C T T Oncomine NM_152770 p.T96M p.T96 Missense_ Hotspot Melanoma Mutation Thyroid C4orf22 4 81504291 SNP C T T Oncomine NM_152770 p.T96M p.T96 Missense_ Hotspot Gland Mutation Papillary Carcinoma Lung C4orf3 4 120221638 SNP C T T Oncomine NM_001001701 p.R18Q p.R18 Missense_ Hotspot Adenocarcinoma Mutation Ductal Breast C4orf3 4 120221638 SNP C C G Oncomine NM_001001701 p.R18P p.R18 Missense_ Hotspot Carcinoma Mutation Lung C4orf3 4 120221638 SNP C T T Oncomine NM_001001701 p.R18Q p.R18 Missense_ Hotspot Adenocarcinoma Mutation Prostate CACNG3 16 24373167 SNP C T T Oncomine NM_006539 p.R311C p.R311 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous CACNG3 16 24372868 SNP C T T Oncomine NM_006539 p.S211F p.S211 Missense_ Hotspot Melanoma Mutation Lung CACNG3 16 24372930 SNP C T T Oncomine NM_006539 p.R232W p.R232 Missense_ Hotspot Adenocarcinoma Mutation Glioblastoma CACNG3 16 24366270 SNP G A A Oncomine NM_006539 p.A138T p.A138 Missense_ Hotspot Mutation Astrocytoma CACNG3 16 24373167 SNP C T T Oncomine NM_006539 p.R311C p.R311 Missense_ Hotspot Mutation Colorectal CACNG3 16 24273772 SNP C T T Oncomine NM_006539 p.A138V p.A138 Missense__ Hotspot Mucinous Mutation Adenocarcinoma Colorectal CACNG3 16 24273771 SNP G A A Oncomine NM_006539 p.A138T p.A138 Missense_ Hotspot Adenocarcinoma Mutation Lung CACNG3 16 24372930 SNP C T T Oncomine NM_006539 p.R232W p.R232 Missense_ Hotspot Adenocarcinoma Mutation Squamous CACNG3 16 24373168 SNP G C C Oncomine NM_006539 p.R311P p.R311 Missense_ Hotspot Cell Lung Mutation Carcinoma Squamous CACNG3 16 24373168 SNP G A A Oncomine NM_006539 p.R311H p.R311 Missense_ Hotspot Cell Lung Mutation Carcinoma Cutaneous CACNG3 16 24372930 SNP C T T Oncomine NM_006539 p.R232W p.R232 Missense_ Hotspot Melanoma Mutation Cutaneous CACNG3 16 24372868 SNP C T T Oncomine NM_006539 p.S211F p.S211 Missense_ Hotspot Melanoma Mutation Cutaneous CCDC61 19 46498687 SNP G A A Oncomine NM_001080402 p.E29K p.E29 Missense_ Hotspot Melanoma Mutation Cutaneous CCDC61 19 46498700 SNP C T T Oncomine NM_001080402 p.S33F p.S33 Missense_ Hotspot Melanoma Mutation Cutaneous CCDC61 19 46498687 SNP G A A Oncomine NM_001080402 p.E29K p.E29 Missense_ Hotspot Melanoma Mutation Prostate CDC27 17 45234367 SNP A A T Oncomine NM_001256 p.S252T p.S252 Missense_ Hotspot Carcinoma Mutation Cutaneous CDC27 17 45234366 SNP G A A Oncomine NM_001256 p.S252F p.S252 Missense_ Hotspot Melanoma Mutation Chromophobe CDC27 17 45234367 SNP A A T Oncomine NM_001256 p.S252T p.S252 Missense_ Hotspot Renal Cell Mutation Carcinoma Cutaneous CNTN5 11 100169975 SNP G A A Oncomine NM_014361 p.E823K p.E823 Missense_ Hotspot Melanoma Mutation Cutaneous CNTN5 11 100170080 SNP G A A Oncomine NM_014361 p.G858R p.G858 Missense_ Hotspot Melanoma Mutation Cutaneous CNTN5 11 99932099 SNP C T T Oncomine NM_014361 p.S379F p.S379 Missense_ Hotspot Melanoma Mutation Cutaneous CNTN5 11 99715827 SNP G A A Oncomine NM_014361 p.R137Q p.R137 Missense_ Hotspot Melanoma Mutation Colorectal CNTN5 11 99221037 SNP G T T Oncomine NM_014361 p.R137L p.R137 Missense_ Hotspot Adenocarcinoma Mutation Colorectal CNTN5 11 99221037 SNP G A A Oncomine NM_014361 p.R137Q p.R137 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous CNTN5 11 99690287 SNP C T T Oncomine NM_014361 p.S23F p.S23 Missense_ Hotspot Melanoma Mutation Cutaneous CNTN5 11 10016997 SNP G A A Oncomine NM_014361 p.E823K p.E823 Missense_ Hotspot Melanoma Mutation Cutaneous CNTN5 11 99932099 SNP C T T Oncomine NM_014361 p.S379F p.S379 Missense_ Hotspot Melanoma Mutation Cutaneous CNTN5 11 10017008 SNP G A A Oncomine NM_014361 p.G858E p.G858 Missense_ Hotspot Melanoma Mutation Cutaneous CNTN5 11 99715827 SNP G A A Oncomine NM_014361 p.R137Q p.R137 Missense_ Hotspot Melanoma Mutation Cutaneous CNTN5 11 51001262 SNP G A A Oncomine NM_014361 p.E681K p.E681 Missense_ Hotspot Melanoma Mutation Cutaneous CNTN5 11 100170080 SNP G A A Oncomine NM_014361 p.G858R p.G858 Missense_ Hotspot Melanoma Mutation Astrocytoma CXCR2 2 219000407 SNP G C C Oncomine NM_001557 p.A295P p.A295 Missense_ Hotspot Endometrial CXCR2 2 218999763 SNP G G A Oncomine NM_001557 p.R80H p.R80 Missense_ Hotspot Endometrioid Mutation Adenocarcinoma Squamous CXCR2 2 218999763 SNP G A A Oncomine NM_001557 p.R80H p.R80 Missense_ Hotspot Cell Lung Mutation Carcinoma Cutaneous CXCR2 2 219000488 SNP C T T Oncomine NM_001557 p.R322C p.R322 Missense_ Hotspot Melanoma Mutation Cutaneous CXCR2 2 219000408 SNP C T T Oncomine NM_001557 p.A295V p.A295 Missense_ Hotspot Melanoma Mutation Cutaneous DCD 12 55039462 SNP C T T Oncomine NM_053283 p.E43K p.E43 Missense_ Hotspot Melanoma Mutation Cutaneous DCD 12 55039462 SNP C T T Oncomine NM_053283 p.E43K p.E43 Missense_ Hotspot Melanoma Mutation Cutaneous DSCR6 21 38390367 SNP G A A Oncomine NM_018962 p.E145K p.E145 Missense_ Hotspot Melanoma Mutation Lung DUX4L2 10 135491125 SNP G A A Oncomine NM_001127386 p.A246T p.A246 Missense_ Hotspot Adenocarcinoma Mutation Lung DUX4L2 10 135491123 SNP G A A Oncomine NM_001127386 p.G245D p.G245 Missense_ Hotspot Adenocarcinoma Mutation Infiltrating DUX4L2 10 135491113 SNP G T T Oncomine NM_001127386 p.A242S p.A242 Missense_ Hotspot Bladder Mutation Urothelial Carcinoma Glioblastoma DUX4L2 10 135491113 SNP G A A Oncomine NM_001127386 p.A242T p.A242 Missense_ Hotspot Mutation Glioblastoma DUX4L2 10 135491125 SNP G A A Oncomine NM_001127386 p.A246T p.A246 Missense_ Hotspot Mutation Glioblastoma DUX4L2 10 135491123 SNP G A A Oncomine NM_001127386 p.G245D p.G245 Missense_ Hotspot Mutation Astrocytoma DUX4L2 10 135491112 SNP C A A Oncomine NM_001127386 p.F241L p.F241 Missense_ Hotspot Mutation Head and DUX4L2 10 135491123 SNP G A A Oncomine NM_001127386 p.A246T p.A246 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Head and DUX4L2 10 135491123 SNP G A A Oncomine NM_001127386 p.G245D p.G245 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Head and DUX4L2 10 135491112 SNP C A A Oncomine NM_001127386 p.F241L p.F241 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Cutaneous DUX4L2 10 135491107 SNP G A A Oncomine NM_001127386 p.A240T p.A240 Missense_ Hotspot Melanoma Mutation Cutaneous DUX4L2 10 135491123 SNP G A A Oncomine NM_001127386 p.G245D p.G245 Missense_ Hotspot Melanoma Mutation Cutaneous DUX4L2 10 135491125 SNP G A A Oncomine NM_001127386 p.A246T p.A246 Missense_ Hotspot Melanoma Mutation Cutaneous DUX 4L2 10 135491113 SNP G A A Oncomine NM_001127386 p.A242T p.A242 Missense_ Hotspot Melanoma Mutation Cutaneous DUX4L2 10 135491112 SNP C A A Oncomine NM_001127386 p.F241L p.F241 Missense_ Hotspot Melanoma Mutation Papillary DUX4L2 10 135491112 SNP C A A Oncomine NM_001127386 p.F241L p.F241 Missense_ Hotspot Renal Cell Mutation Carcinoma Thyroid DUX 4L2 10 135491125 SNP G A A Oncomine NM_001127386 p.A246T p.A246 Missense_ Hotspot Gland Mutation Papillary Carcinoma Thyroid DUX4L2 10 135491107 SNP G A A Oncomine NM_001127386 p.A240T p.A240 Missense_ Hotspot Gland Mutation Papillary Carcinoma Thyroid DUX4L2 10 135491113 SNP G A A Oncomine NM_001127386 p.A242T p.A242 Missense_ Hotspot Gland Mutation Papillary Carcinoma Thyroid DUX4L2 10 135491123 SNP G A A Oncomine NM_001127386 p.G245D p.G245 Missense_ Hotspot Gland Mutation Papillary Carcinoma Cutaneous EDDM3A 14 21216002 SNP G A A Oncomine NM_006683 p.R88Q p.R88 Missense_ Hotspot Melanoma Mutation Glioblastoma EDDM3A 14 21216002 SNP G A A Oncomine NM_006683 p.R88Q p.R88 Missense_ Hotspot Mutation Colorectal EDDM3A 14 20285842 SNP G G A Oncomine NM_006683 p.R88Q p.R88 Missense_ Hotspot Mucinous Mutation Adenocarcinoma Cutaneous EDDM3A 14 21216002 SNP G A A Oncomine NM_006683 p.R88Q p.R88 Missense_ Hotspot Melanoma Mutation Ductal Breast ENDOU 12 48110712 SNP G G A Oncomine NM_006025 p.P10L3 p.P130 Missense_ Hotspot Carcinoma Mutation Endometrial ENDOU 12 48110712 SNP G G A Oncomine NM_006025 p.P130L p.P130 Missense_ Hotspot Endometrioid Mutation Adenocarcinoma Cutaneous ENDOU 12 48110713 SNP G C C Oncomine NM_006025 p.P130A p.P130 Missense_ Hotspot Melanoma Mutation Colorectal ERAS X 48572767 SNP C T T Oncomine NM_181532 p.A97V p.A97 Missense_ Hotspot Adenocarcinoma Mutation Endometrial ERAS X 48687822 SNP G G A Oncomine NM_181532 p.A97T p.A97 Missense_ Hotspot Endometrioid Mutation Adenocarcinoma Lung ERAS X 48687822 SNP G A A Oncomine NM_181532 p.A97T p.A97 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous FABP1 2 88425751 SNP C T T Oncomine NM_001443 p.E62K p.E62 Missense_ Hotspot Melanoma Mutation Cutaneous FABP1 2 88425751 SNP C T T Oncomine NM_001443 p.E62K p.E62 Missense_ Hotspot Melanoma Mutation Medulloblastoma FAM22F 9 97082793 DEL AGA * * Oncomine NM_017561 p.S691_in_frame_del p.S691_in_frame_del In_Frame_Del Hotspot Cervical FAM22F 9 97082793 SNP C G G Oncomine NM_017561 p.K355N p.K355 Missense_ Hotspot Squamous Mutation Cell Carcinoma Colorectal FAM22F 9 96122614 SNP C G G Oncomine NM_017561 p.K355N p.K355 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous FAM22F 9 97080945 DEL AGA — — Oncomine NM_017561 p.S691_in_frame_del p.S691_in_frame_del In_Frame_Del Hotspot Melanoma Prostate FAM22F 9 97080945 DEL AGA — — Oncomine NM_017561 p.S691_in_frame_del p.S691_in_frame_del In_Frame_Del Hotspot Adenocarcinoma Thyroid FAM22F 9 97080945 DEL AGA — — Oncomine NM_017561 p.S691_in_frame_del p.S691_in_frame_del In_Frame_Del Hotspot Gland Carcinoma, NOS Ductal Breast FBXW8 12 117465850 SNP G G A Oncomine NM_012174 p.R491H p.R491 Missense_ Hotspot Carcinoma Mutation Colorectal FBXW8 12 115950233 SNP G A A Oncomine NM_012174 p.R491H p.R491 Missense_ Hotspot Adenocarcinoma Mutation Head and FBXW8 12 117465849 SNP C T T Oncomine NM_012174 p.R491C p.R491 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Squamous FBXW8 12 117465849 SNP C T T Oncomine NM_012174 p.R491C p.R491 Missense_ Hotspot Cell Lung Mutation Carcinoma Cutaneous FBXW8 12 117465849 SNP C T T Oncomine NM_012174 p.R491C p.R491 Missense_ Hotspot Melanoma Mutation Glioblastoma FHL3 1 38463709 SNP G A A Oncomine NM_004468 p.P143S p.P143 Missense_ Hotspot Mutation Lung FH3L 1 38463709 SNP G A A Oncomine NM_004468 p.P143S p.P143 Missense_ Hotspot Adenocarcinona Mutation Thyroid FHL3 1 38463709 SNP G C C Oncomine NM_004468 p.P143A p.P143 Missense_ Hotspot Gland Mutation Papillary Carcinoma Colorectal GGT1 2 23340828 SNP G A A Oncomine NM_005265 p.G84S p.G84 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous GK2 4 80327859 SNP C G G Oncomine NM_033214 p.R499P p.R499 Missense_ Hotspot Melanoma Mutation Cutaneous GK2 4 80328367 SNP G A A Oncomine NM_033214 p.R330C p.R330 Missense_ Hotspot Melanoma Mutation Cutaneous GK2 4 80327860 SNP G A A Oncomine NM_033214 p.R499C p.R499 Missense_ Hotspot Melanoma Mutation Lung GK2 4 80328367 SNP G A A Oncomine NM_033214 p.R330C p.R330 Missense_ Hotspot Adenocarcinoma Mutation Glioblastoma GK2 4 80328891 SNP C A A Oncomine NM_033214 p.R155L p.R155 Missense_ Hotspot Mutation Colorectal GK2 4 80547121 SNP G A A Oncomine NM_033214 p.R420C p.R420 Missense_ Hotspot Adenocarcinoma Mutation Endometrial GK2 4 80328892 SNP G G A Oncomine NM_033214 p.R155C p.R155 Missense_ Hotspot Endometrioid Mutation Adenocarcinoma Head and GK2 4 80327860 SNP G A A Oncomine NM_033214 p.R499C p.R499 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Lung GK2 4 80328679 SNP G A A Oncomine NM_033214 p.P226S p.P226 Missense_ Hotspot Adenocarcinoma Mutation Squamous GK2 4 80328678 SNP G A A Oncomine NM_033214 p.P226L p.P226 Missense_ Hotspot Cell Lung Mutation Carcinoma Cutaneous GK2 4 80328892 SNP G A A Oncomine NM_033214 p.R155C p.R155 Missense_ Hotspot Melanoma Mutation Cutaneous GK2 4 80328367 SNP G A A Oncomine NM_033214 p.R330C p.R330 Missense_ Hotspot Melanoma Mutation Cutaneous GK2 4 80327860 SNP G A A Oncomine NM_033214 p.R499C p.R499 Missense_ Hotspot Melanoma Mutation Cutaneous GK2 4 80328097 SNP G A A Oncomine NM_033214 p.R420C p.R420 Missense_ Hotspot Melanoma Mutation Cutaneous GK2 4 80328679 SNP G A A Oncomine NM_033214 p.P226S p.P226 Missense_ Hotspot Melanoma Mutation Glioblastoma GOLGA6L10 15 83014132 SNP C G G Oncomine NM_001164465 p.E151Q p.E151 Missense_ Hotspot Mutation Ductal Breast GOLGA6L10 15 83014132 SNP C C G Oncomine NM_001164465 p.E151Q p.E151 Missense_ Hotspot Carcinoma Mutation Head and GOLGA6L10 15 83014132 SNP C G G Oncomine NM_001164465 p.E151Q p.E151 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Clear Cell GOLGA6L10 15 83014132 SNP C G G Oncomine NM_001164465 p.E151Q p.E151 Missense_ Hotspot Renal Cell Mutation Carcinoma Thyroid GOLGA6L10 15 83014132 SNP C G G Oncomine NM_001164465 p.E151Q p.E151 Missense_ Hotspot Gland Mutation Papillary Carcinoma Cutaneous GPX7 1 53072530 SNP C T T Oncomine NM_015696 p.R105C p.R105 Missense_ Hotspot Melanoma Mutation Head and GPX7 1 53072531 SNP G T T Oncomine NM_015696 p.R105L p.R105 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Lung GPX7 1 53072531 SNP G A A Oncomine NM_015696 p.R105H p.R105 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous GTSF1 12 54858877 SNP G A A Oncomine NM_144594 p.P31S p.P31 Missense_ Hotspot Melanoma Mutation Cutaneous GTSF1 12 548588 SNP G A A Oncomine NM_144594 p.P31S p.P31 Missense_ Hotspot Melanoma Mutation Head and H3F3A 1 226252059 SNP C T T Oncomine NM_002107 p.R3C p.R3 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Astrocytoma H3F3A 1 226252059 SNP C T T Oncomine NM_002107 p.R3C p.R3 Missense_ Hotspot Mutation Cervical H3F3A 1 226252059 SNP C T T Oncomine NM_002107 p.R3C p.R3 Missense_ Hotspot Squamous Mutation Cell Carcinoma Small Cell HDDC2 6 125661566 SNP C G G Oncomine NM_016063 p.R101P p.R101 Missene_ Hotspot Lung Mutation Carcinoma Small Cell HDDC2 6 125619867 SNP C G G Oncomine NM_016063 p.R101P p.R101 Missense_ Hotspot Lung Mutation Carcinoma Head and HDDC2 6 125619867 SNP C T T Oncomine NM_016063 p.R101Q p.R101 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Cutaneous HEXDC 17 80400154 SNP A C C Oncomine NM_173620 p.T482P p.T482 Missense_ Hotspot Melanoma Mutation Squamous HEXDC 17 80400154 SNP A C C Oncomine NM_173620 p.T482P p.T482 Missense_ Hotspot Cell Lung Mutation Carcinoma Clear Cell HEXDC 17 80400154 SNP A C C Oncomine NM_173620 p.T482P p.T482 Missense_ Hotspot Renal Cell Mutation Carcinoma Small Cell HIST1H4C 6 26212357 SNP G C C Oncomine NM_003542 p.R68P p.R68 Missense_ Hotspot Lung Mutation Carcinoma Head and HIST1H4C 6 26104378 SNP G C C Oncomine NM_003542 p.R68P p.R68 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Cutaneous HNRNPCL1 1 12907971 SNP C T T Oncomine NM_001013631 p.D58N p.D58 Missense_ Hotspot Melanoma Mutation Melanoma HNRNPCL1 1 12907847 SNP C T T Oncomine NM_001013631 p.R99Q p.R99 Missense_ Hotspot Mutation Colorectal HNRNPCL1 1 12830231 SNP G A A Oncomine NM_001013631 p.R167W p.R167 Missense_ Hotspot Adenocarcinoma Mutation Endometrial HNRNPCL1 1 12907644 SNP G G A Oncomine NM_001013631 p.R167W p.R167 Missense_ Hotspot Endometrioid Mutation Adenocarcinoma Gastric HNRNPCL1 1 12907847 SNP C T T Oncomine NM_001013631 p.R99Q p.R99 Missense_ Hotspot Adenocarcinoma Mutation Lung HNRNPCL1 1 12907643 SNP C A A Oncomine NM_001013631 p.R167L p.R167 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous HNRNPCL1 1 12907847 SNP C T T Oncomine NM_001013631 p.R99Q p.R99 Missense_ Hotspot Melanoma Mutation Cutaneous HNRNPCL1 1 12907865 SNP C T T Oncomine NM_001013631 p.G93E p.G93 Missense_ Hotspot Melanoma Mutation Cutaneous HNRNPCL1 1 12907971 SNP C T T Oncomine NM_001013631 p.D58N p.D58 Missense_ Hotspot Melanoma Mutation Prostate HRCT1 9 35906559 SNP A C C Oncomine NM_001039792 p.H92P p.H92 Missense_ Hotspot Adenocarcinoma Mutation Glioblastoma HRCT1 9 35906348 DEL CTG — — Oncomine NM_001039792 p.L22_in_frame_del p.L22_in_frame_del In_Frame_Del Hotspot Ductal Breast HRCT1 9 35906348 DEL CTG CTG — Oncomine NM_001039792 p.L22_in_frame_del p.L22_in_frame_del In_Frame_Del Hotspot Carcinoma Cervical HRCT1 9 35906559 SNP A C C Oncomine NM_001039792 p.H92P p.H92 Missense_ Hotspot Squamous Mutation Cell Carcinoma Gastric HRCT1 9 35906584 DEL CCA — — Oncomine NM_001039792 p.L100_in_frame_del pL100_in_frame_del In_Frame_Del Hotspot Adenocarcinoma Cutaneous HRCT1 9 35906348 DEL CTG — — Oncomine NM_001039792 p.L22_in_frame_del p.L22_in_frame_del In_Frame_Del Hotspot Melanoma Cutaneous HRCT1 9 35906559 SNP A C C Oncomine NM_001039792 p.H92P p.H92 Missense_ Hotspot Melanoma Mutation Papillary HRCT1 9 35906584 DEL CCA — — Oncomine NM_001039792 p.L100_in_frame_del p.L100_in_frame_del In_Frame_Del Hotspot Renal Cell Carcinoma Papillary HRCT1 9 35906559 SNP A C C Oncomine NM_001039792 p.H92P p.H92 Missense_ Hotspot Renal Cell Mutation Carcinoma Thyroid HRCT1 9 35906584 DEL CCA — — Oncomine NM_001039792 p.L100_in_frame_del p.L100_in_frame_del In_Frame_Del Hotspot Gland Carcinoma, NOS Colorectal IL3 5 131425967 SNP G A A Oncomine NM_000588 p.A90T p.A90 Missense_ Hotspot Adenocarcinoma Mutation Gastric IL3 5 131398068 SNP G A A Oncomine NM_000588 p.A90T p.A90 Missense_ Hotspot Adenocarcinoma Mutation Pancreatic JAM3 11 134014849 SNP G A G Oncomine NM_032801 p.R191H p.R191 Missense_ Hotspot Ductal Mutation Adenocarcinoma Lobular JAM3 11 134014849 SNP G G A Oncomine NM_032801 p.R191H p.R191 Missense_ Hotspot Breast Mutation Carcinoma Gastric JAM3 11 134014848 SNP C T T Oncomine NM_032801 p.R191C p.R191 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous KCNK9 8 140631316 SNP C T T Oncomine NM_016601 p.D104N p.D104 Missense_ Hotspot Melanoma Mutation Endometrial KCNK9 8 140630833 SNP C C T Oncomine NM_016601 p.A265T p.A265 Missense_ Hotspot Endometrioid Mutation Adenocarcinoma Lung KCNK9 8 140630832 SNP G A A Oncomine NM_016601 p.A265V p.A265 Missense_ Hotspot Adenocarcinoma Mutation Squamous KCNK9 8 140630833 SNP C T T Oncomine NM_016601 p.A265T p.A265 Missense_ Hotspot Cell Lung Mutation Carcinoma Cutaneous KCNK9 8 140631316 SNP C T T Oncomine NM_016601 p.D104N p.D104 Missense_ Hotspot Melanoma Mutation Glioblastoma KLK6 19 51466671 SNP C T T Oncomine NM_002774 p.R111H p.R111 Missense_ Hotspot Mutation Colorectal KLK6 19 56158484 SNP G A A Oncomine NM_002774 p.R111C p.R111 Missense_ Hotspot Mucinous Mutation Adenocarcinoma Endometrial KLK6 19 51466671 SNP C C T Oncomine NM_002774 p.R111H p.R111 Missense_ Hotspot Endometrioid Mutation Adenocarcinoma Cutaneous KLK6 19 51462556 SNP G A A Oncomine NM_002774 p.P200L p.P200 Missense_ Hotspot Melanoma Mutation Prostate KLK6 19 51462556 SNP G A A Oncomine NM_002774 p.P200L p.P200 Missense_ Hotspot Adenocarcinoma Mutatopm Clear Cell KLK6 19 51462556 SNP G A A Oncomine NM_002774 p.P200L p.P200 Missense_ Hotspot Renal Cell Mutation Carcinoma Colorectal KRTAP12-4 21 44898950 SNP T G G Oncomine NM_198698 p.T4P p.T4 Missense_ Hotspot Adenocarcinoma Mutation Ovarian KRTAP12-4 21 44898949 SNP G G A Oncomine NM_198698 p.T4I p.T4 Missense_ Hotspot Serous Mutation Adenocarcinoma Cutaneous KRTAP4-11 17 39274424 SNP G C C Oncomine NM_033059 p.S48R p.S48 Missense_ Hotspot Melanoma Mutation Cutaneous KRTAP4-11 17 39274206 SNP C T T Oncomine NM_033059 p.R121K p.R121 Missense_ Hotspot Melanoma Mutation Lung KRTAP4-11 17 39274150 SNP T A A Oncomine NM_033059 p.S140C p.S140 Missense_ Hotspot Adenocarcinoma Mutation Lung KRTAP4-11 17 39274206 SNP C T T Oncomine NM_033059 p.R121K p.R121 Missense_ Hotspot Adenocarcinoma Mutation Glioblastoma KRTAP4-11 17 39274424 SNP G C C Oncomine NM_033059 p.S48R p.S48 Missense_ Hotspot Oligodendroglioma KRTAP4-11 17 39274087 SNP G C C Oncomine NM_033059 p.L161V p.L161 Missense_ Hotspot Mutation Ductal Breast KRTAP4-11 17 39274087 SNP G G C Oncomine NM_033059 p.L161V p.L161 Missense_ Hotspot Carcinoma Mutation Cervical KRTAP4-11 17 39274424 SNP G C C Oncomine NM_033059 p.548R p.548 Missense_ Hotspot Squamous Mutation Cell Carcinoma Cervical KRTAP4-11 17 39274087 SNP G C C Oncomine NM_033059 p.L161V p.L161 Missense_ Hotspot Squamous Mutation Cell Carcinoma Cervical KRTAP4-11 17 39274150 SNP T A A Oncomine NM_033059 p.S140C p.S140 Missense_ Hotspot Squamous Mutation Cell Carcinoma Head and KRTAP4-11 17 39274424 SNP G C C Oncomine NM_033059 p.S48R p.S48 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Head and KRTAP4-11 17 39274206 SNP C T T Oncomine NM_033059 p.R121K p.R121 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Head and KRTAP4-11 17 39274087 SNP G C C Oncomine NM_033059 p.L161V p.L161 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Head and KRTAP4-11 17 39274150 SNP T A A Oncomine NM_033059 p.S140C p.S140 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Head and KRTAP4-11 17 39274291 SNP T C C Oncomine NM_033059 p.M93V p.M93 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Head and KRTAP4-11 17 39274416 SNP C T T Oncomine NM_033059 p.R51K p.R51 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Lung KRTAP4-11 17 39274424 SNP G C C Oncomine NM_033059 p.548R p.548 Missense_ Hotspot Adenocarcinoma Mutation Lung KRTAP4-11 17 39274206 SNP C T T Oncomine NM_033059 p.R121K p.R121 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous KRTAP4-11 17 39274150 SNP T A A Oncomine NM_033059 p.5140C p.S140 Missense_ Hotspot Melanoma Mutation Cutaneous KRTAP4-11 17 39274206 SNP C T T Oncomine NM_033059 p.R121K p.R121 Missense_ Hotspot Melanoma Mutation Cutaneous KRTAP4-11 17 39274424 SNP G C C Oncomine NM_033059 p.S48R p.S48 Missense_ Hotspot Melanoma Mutation Cutaneous KRTAP4-11 17 39274087 SNP G C C Oncomine NM_033059 p.L161V p.L161 Missense_ Hotspot Melanoma Mutatopm Cutaneous KRTAP4-11 17 39274416 SNP C T T Oncomine NM_033059 p.R51K p.R51 Missense_ Hotspot Melanoma Mutation Clear Cell KRTAP4-11 17 39274291 SNP T C C Oncomine NM_033059 p.M93V p.M93 Missense_ Hotspot Renal Cell Mutation Carcinoma Clear Cell KRTAP4-11 17 39274206 SNP C T T Oncomine NM_033059 p.R121K p.R121 Missense_ Hotspot Renal Cell Mutation Carcinoma Clear Cell KRTAP4-11 17 39274150 SNP T A A Oncomine NM_033059 p.S140C p.S140 Missense_ Hotspot Renal Cell Mutation Carcinoma Papillary KRTAP4-11 17 39274087 SNP G C C Oncomine NM_033059 p.L161V p.L161 Missense_ Hotspot Renal Cell Mutation Carcinoma Thyroid KRTAP4-11 17 39274424 SNP G C C Oncomine NM_033059 p.S48R p.S48 Missense_ Hotspot Gland Mutation Papillary Carcinoma Papillary KRTAP4-7 17 39240900 SNP T G G Oncomine NM_033061 p.L148V p.L148 Missense_ Hotspot Renal Cell Mutation Carcinoma Cutaneous LAD1 1 201354881 SNP C T T Oncomine NM_005558 p.R360Q p.R360 Missense_ Hotspot Melanoma Mutation Cutaneous LAD1 1 201352246 SNP C T T Oncomine NM_005558 p.E448K p.E448 Missense_ Hotspot Melanoma Mutation Clear Cell LAD1 1 201354881 SNP C A A Oncomine NM_005558 p.R360L p.R360 Missense_ Hotspot Renal Cell Mutation Carcinoma Melanoma LELP1 1 153177244 SNP C T T Oncomine NM_001010857 p.P21S p.P21 Missense_ Hotspot Mutation Cutaneous LELP1 1 153177437 SNP C T T Oncomine NM_01010857 p.S85F p.S85 Missense_ Hotspot Melanoma Mutation Cutaneous LELP1 1 153177245 SNP C T T Oncomine NM_001010857 p.P21L p.P21 Missense_ Hotspot Melanoma Mutation Cutaneous LELP1 1 153177244 SNP C T T Oncomine NM_001010857 p.P21S p.P21 Missense_ Hotspot Melanoma Mutation Cutaneous LOC100509575 X 47972582 SNP G A A Oncomine NM_001205103 p.R96H p.R96 Missense_ Hotspot Melanoma Mutation Lobular LOC100509575 X 47972582 SNP G G A Oncomine NM_001205103 p.R96H p.R96 Missense_ Hotspot Breast Mutation Carcinoma Endometrial LOC100509575 X 47972581 SNP C C T Oncomine NM_001205103 p.R96C p.R96 Missense_ Hotspot Endometrioid Mutation Adenocarcinoma Glioblastoma MUC4 3 195516064 SNP C T T Oncomine NM_018406 p.R796Q p.R796 Missense_ Hotspot Mutation Ductal Breast MUC4 3 195516064 SNP C C T Oncomine NM_018406 p.R796Q p.R796 Missense_ Hotspot Carcinoma Lung MUC4 3 195516064 SNP C T T Oncomine NM_018406 p.R796Q p.R796 Missense_ Hotspot Adenocarcinoma Mutation Glioblastoma NAB2 12 57485446 SNP T C C Oncomine NM_005967 p.F208L p.F208 Missense_ Hotspot Mutation Oligodendroglioma NAB2 12 57485446 SNP P T C Oncomine NM_005967 p.F208L p.F208 Missense_ Hotspot Mutation Head and NAB2 12 57485446 SNP T C C Oncomine NM_005967 p.F208L p.F208 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Lung NAB2 12 57485446 SNP T C C Oncomine NM_005967 p.F208L p.F208 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous NAB2 12 57485446 SNP T C C Oncomine NM_005967 p.F208L p.F208 Missense_ Hotspot Melanoma Mutation Mutation Glioblastoma NBPF10 1 145324371 SNP T C C Oncomine NM_001039703 p.V1189A p.V1189 Missense_ Hotspot Mutation Astrocytoma NBPF10 1 145360584 SNP G A A Oncomine NM_001039703 p.G3070E p.G3070 Missense_ Hotspot Cutaneous NBPF10 1 145360584 SNP G A A Oncomine NM_001039703 p.G3070E p.G3070 Missense_ Hotspot Melanoma Mutation Cutaneous NSFL1C 20 1426360 SNP G A A Oncomine NM_016143 p.R301W p.R301 Missense_ Hotspot Melanoma Mutation Colorectal NSFL1C 20 1374360 SNP G A A Oncomine NM_016143 p.R301W p.R301 Missense_ Hotspot Adenocarcinoma Mutation Endometrial NSFL1C 20 1426360 SNP G G A Oncomine NM_016143 p.R301W p.R301 Missense_ Hotspot Endometrioid Mutation Adenocarcinoma Head and NSFL1C 20 1426360 SNP G A A Oncomine NM_016143 p.R301W p.R301 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Medulloblastoma OBP2B 9 136081795 SNP A G G Oncomine NM_014581 p.S133P p.S133 Missense_ Hotspot Mutation Head and OBP2B 9 136081795 SNP A G G Oncomine NM_014581 p.S133P p.S133 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Lung OBP2B 9 136081795 SNP A G G Oncomine NM_014581 p.S133P p.S133 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous OR2L13 1 248262729 SNP C A A Oncomine NM_175911 p.P18T p.P18 Missense_ Hotspot Melanoma Mutation Cutaneous OR2L13 1 248263173 SNP C T T Oncomine NM_175911 p.P166S p.P166 Missense_ Hotspot Melanoma Mutation Lung OR2L13 1 248263401 SNP A G G Oncomine NM_175911 p.T242A p.T242 Missense_ Hotspot Adenocarcinoma Mutation Small Cell OR2L13 1 248262832 SNP C A A Oncomine NM_175911 p.P52H p.P52 Missense_ Hotspot Lung Mutation Carcinoma Head and OR2L13 1 248262831 SNP C T T Oncomine NM_175911 p.P52S p.P52 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Lung OR2L13 1 248263401 SNP A T T Oncomine NM_175911 p.T242S p.T242 Missense_ Hotspot Adenocarcinoma Mutation Lung OR2L13 1 248263401 SNP A G G Oncomine NM_175911 p.T242A p.T242 Missense_ Hotspot Adenocarcinoma Mutation Squamous OR2L13 1 248262831 SNP C T T Oncomine NM_175911 p.P525 p.P52 Missense_ Hotspot Cell Lung Mutation Carcinoma Cutaneous OR2L13 1 248263371 SNP G A A Oncomine NM_175911 p.G232R p.G232 Missense_ Hotspot Melanoma Mutation Cutaneous OR2 L13 1 248263171 SNP C T T Oncomine NM_175911 p.P166L p.P166 Missense_ Hotspot Melanoma Mutation Cutaneous OR2 L13 1 248263173 SNP C T T Oncomine NM_175911 p.P166S p.P166 Missense_ Hotspot Melanoma Mutation Cutaneous OR2L13 1 248262730 SNP C T T Oncomine NM_175911 p.P18L p.P18 Missense_ Hotspot Melanoma Mutation Cutaneous OR2L13 1 248262721 SNP C A A Oncomine NM_175911 p.P18T p.P18 Missense_ Hotspot Melanoma Mutation Ovarian OR2L13 1 246329995 SNP G A A Oncomine NM_175911 p.G232E p.G232 Missense_ Hotspot Serous Mutation Adenocarcinoma Colorectal OR2T27 1 246880778 SNP C T T Oncomine NM_001001824 p.D11N p.D11 Missense_ Hotspot Adenocarcinoma Mutation Endometrial OR2T27 1 248813822 SNP G G A Oncomine NM_001001824 p.R122C p.R122 Missense_ Hotspot Serous Mutation Adenocarcinoma Gastric OR2T27 1 248813821 SNP C T T Oncomine NM_001001824 p.R122H p.R122 Missense_ Hotspot Adenocarcinoma Mutation Gastric OR2T27 1 248813773 SNP C T T Oncomine NM_001001824 p.R138H p.R138 Missense_ Hotspot Adenocarcinoma Mutation Head and OR2T27 1 248813773 SNP C G G Oncomine NM_001001824 p.R138P p.R138 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Lung OR2T27 1 248814155 SNP C A A Oncomine NM_001001824 p.D11Y p.D11 Missense_ Hotspot Adenocarcinoma Mutation Lung OR2T27 1 248813773 SNP C A A Oncomine NM_001001824 p.R138L p.R138 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous OR2Z1 19 8841458 SNP C T T Oncomine NM_001004699 p.S23L p.S23 Missense_ Hotspot Melanoma Mutation Glioblastoma OR2Z1 19 8841802 SNP C T T Oncomine NM_001004699 p.R138C p.R138 Missense_ Hotspot Mutation Gastric OR2Z1 19 8841802 SNP C T T Oncomine NM_001004699 p.R138C p.R138 Missense_ Hotspot Adenocarcinoma Mutation Lung OR2Z1 19 8841802 SNP C T T Oncomine NM_001004699 p.R138C p.R138 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous OR2Z1 19 8841458 SNP C T T Oncomine NM_001004699 p.S23L p.S23 Missense_ Hotspot Melanoma Mutation Cutaneous OR2Z1 19 8841889 SNP C T T Oncomine NM_001004699 p.P167S p.P167 Missense_ Hotspot Melanoma Mutation Cutaneous OR4E2 14 22133748 SNP G A A Oncomine NM_001001912 p.G151E p.G151 Missense_ Hotspot Melanoma Mutation Cutaneous OR4E2 14 22133973 SNP G A A Oncomine NM_001001912 p.R226Q p.R226 Missense_ Hotspot Melanoma Mutation Cutaneous OR4E2 14 22133747 SNP G A A Oncomine NM_001001912 p.G151R p.G151 Missense_ Hotspot Melanoma Mutation Cutaneous OR4E2 14 22133748 SNP G A A Oncomine NM_001001912 p.G151E p.G151 Missense_ Hotspot Melanoma Mutation Cutaneous OR51B2 11 5345263 SNP C T T Oncomine NM_033180 p.E89K p.E89 Missense_ Hotspot Melanoma Mutation Cutaneous OR51B2 11 5345040 SNP G A A Oncomine NM_033180 p.S163L p.S163 Missense_ Hotspot Melanoma Mutation Lung OR51B2 11 5344773 SNP G T T Oncomine NM_033180 p.T252K p.T252 Missense_ Hotspot Adenocarcinoma Mutation Glioblastoma OR51B2 11 5344773 SNP G A A Oncomine NM_033180 p.T2521 p.T252 Missense_ Hotspot Mutation Head and OR51B2 11 5344774 SNP T C C Oncomine NM_033180 p.T252A p.T252 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Squamous OR51B2 11 5345101 SNP C T T Oncomine NM_033180 p.G143R p.G143 Missense_ Hotspot Cell Lung Mutation Carcinoma Squamous OR51B2 11 5345100 SNP C A A Oncomine NM_033180 p.G143V p.G143 Missense_ Hotspot Cell Lung Mutation Carcinoma Cutaneous OR51B2 11 5345163 SNP C T T Oncomine NM_033180 p.E89K p.E89 Missense_ Hotspot Melanoma Mutation Cutaneous OR51B2 11 5345040 SNP G A A Oncomine NM_033180 p.S163L p.S163 Missense__ Hotspot Melanoma Mutation Cutaneous OR51B2 11 5345100 SNP C T T Oncomine NM_033180 p.G143E p.G143 Missense_ Hotspot Melanoma Mutation Glioblastoma OR52A1 11 5172692 SNP C T T Oncomine NM_012375 p.R303H p.R303 Missense_ Hotspot Mutation Cutaneous OR52A1 11 5172911 SNP G A A Oncomine NM_012375 p.R230C p.R230 Missense_ Hotspot Melanoma Mutation Cutaneous OR52A1 11 5172693 SNP G A A Oncomine NM_012375 p.R303C p.R303 Missense_ Hotspot Melanoma Mutation Prostate OR52A1 11 5172911 SNP C T T Oncomine NM_012375 p.R230H p.R230 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous OR5AN1 11 59132584 SNP C T T Oncomine NM_001004729 p.S218F p.S218 Missense_ Hotspot Melanoma Mutation Cutaneous OR6T1 11 123814182 SNP G A A Oncomine NM_001005187 p.R122C p.R122 Missense_ Hotspot Melanoma Mutation Ductal Breast OR6T1 11 123813896 SNP G G T Oncomine NM_001005187 p.S217Y p.S217 Missense_ Hotspot Carcinoma Mutation Colorectal OR6T1 11 123318974 SNP C C T Oncomine NM_001005187 p.R261H p.R261 Missense_ Hotspot Adenocarcinoma Mutation Colorectal OR6T1 11 123319221 SNP G A A Oncomine NM_001005187 p.R179C p.R179 Missense_ Hotspot Mucinous Mutation Adenocarcinoma Colorectal OR6T1 11 123319106 SNP G T T Oncomine NM_001005187 p.S217Y p.S217 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous OR6T1 11 123813765 SNP G A A Oncomine NM_001005187 p.R261C p.R261 Missense_ Hotspot Melanoma Mutation Cutaneous OR6T1 11 123814011 SNP G A A Oncomine NM_001005187 p.R179C p.R179 Missense_ Hotspot Melanoma Mutation Cutaneous OR6T1 11 123813896 SNP G A A Oncomine NM_001005187 p.S217F p.S217 Missense_ Hotspot Melanoma Mutation Cutaneous OR6T1 11 123814182 SNP G A A Oncomine NM_001005187 p.R122C p.R122 Missense_ Hotspot Melanoma Mutation Clear Cell OR6T1 11 123814182 SNP G T T Oncomine NM_001005187 p.R122S p.R122 Missense_ Hotspot Renal Cell Mutation Carcinoma Cutaneous OTUD5 X 48792073 SNP C T T Oncomine NM_017602 p.R274Q p.R274 Missense_ Hotspot Melanoma Mutation Colorectal OTUD5 X 48668111 SNP G A A Oncomine NM_017602 p.R412W p.R412 Missense_ Hotspot Adenocarcinoma Mutation Colorectal OTUD5 X 48677018 SNP G A A Oncomine NM_017602 p.R274W p.R274 Missense_ Hotspot Adenocarcinoma Mutation Endometrial OTUD5 X 48792074 SNP G G A Oncomine NM_017602 p.R274W p.R274 Missense_ Hotspot Endometrioid Mutation Adenocarcinoma Endometrial OTUD5 X 48783167 SNP G G A Oncomine NM_017602 p.R412W p.R412 Missense_ Hotspot Endometrioid Mutation Adenocarcinoma Lung OTUD5 X 48783166 SNP C A A Oncomine NM_017602 p.R412L p.R412 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous OXA1L 14 23235902 SNP C T T Oncomine NM_005015 p.P58S p.P58 Missense_ Hotspot Melanoma Mutation Cutaneous OXA1L 14 23235899 SNP C T T Oncomine NM_005015 p.L57F p.L57 Missense_ Hotspot Melanoma Mutation Cutaneous OXA1L 14 23235902 SNP C T T Oncomine NM_005015 p.P58S p.P58 Missense_ Hotspot Melanoma Mutation Cervical PBX2 6 32155509 SNP T A A Oncomine NM_002586 p.Y262F p.Y262 Missense_ Hotspot Squamous Mutation Cell Carcinoma Gastric PBX2 6 32155509 SNP T A A Oncomine NM_002586 p.Y262F p.Y262 Missense_ Hotspot Adenocarcinoma Mutation Head and PBX2 6 32155509 SNP T A A Oncomine NM_002586 p.Y262F p.Y262 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Squamous PBX2 6 32155509 SNP T A A Oncomine NM_002586 p.Y262F p.Y262 Missense_ Hotspot Cell Lung Mutation Carcinoma Clear Cell PBX2 6 32155509 SNP T A A Oncomine NM_002586 p.Y262F p.Y262 Missense_ Hotspot Renal Cell Mutation Carcinoma Prostate PDHA2 4 96761513 SNP G A A Oncomine NM_005390 p.R71H p.R71 Missense_ Hotspot Adenocarcinoma Mutation Melanoma PDHA2 4 96761738 SNP G A A Oncomine NM_005390 p.G146E p.G146 Missense_ Hotspot Mutation Cutaneous PDHA2 4 96761737 SNP G A A Oncomine NM_005390 p.G146R p.G146 Missense_ Hotspot Melanoma Mutation Glioblastoma PDHA2 4 96761557 SNP C T T Oncomine NM_005390 p.R86C p.R86 Missense_ Hotspot Mutation Colorectal PDHA2 4 96980580 SNP C T T Oncomine NM_005390 p.R86C p.R86 Missense_ Hotspot Adenocarcinoma Mutation Endometrial PDHA2 4 96761738 SNP G G A Oncomine NM_005390 p.G146E p.G146 Missense_ Hotspot Serous Mutation Adenocarcinoma Lung PDHA2 4 96761513 SNP G A A Oncomine NM_005390 p.R71H p.R71 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous PDHA2 4 96761854 SNP G A A Oncomine NM_005390 p.D185N p.D185 Missense_ Hotspot Melanoma Mutation Cutaneous PDHA2 4 96761738 SNP G A A Oncomine NM_005390 p.G146E p.G146 Missense_ Hotspot Melanoma Mutation Thyroid PDHA2 4 96761513 SNP G A A Oncomine NM_005390 p.R71H p.R71 Missense_ Hotspot Gland Mutation Carcinoma, NOS Thyroid PDHA2 4 96761557 SNP C T T Oncomine NM_005390 p.R86C p.R86 Missense_ Hotspot Gland Mutation Papillary Carcinoma Lung POTEC 18 14543019 SNP T C C Oncomine NM_001137671 p.M43V p.M43 Missense_ Hotspot Adenocarcinoma Mutation Glioblastoma POTEC 18 14543019 SNP T C C Oncomine NM_001137671 p.M43V p.M43 Missense_ Hotspot Mutation Astrocytoma POTEC 18 14513734 SNP C T T Oncomine NM_001137671 p.G487E p.G487 Missense_ Hotspot Mutation Head and POTEC 18 14513734 SNP C T T Oncomine NM_001137671 p.G487E p.G487 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Head and POTEC 18 14543019 SNP T C C Oncomine NM_001137671 p.M43V p.M43 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Lung POTEC 18 14513734 SNP C T T Oncomine NM_001137671 p.G487E p.G487 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous POTEC 18 14543019 SNP T C C Oncomine NM_001137671 p.M43V p.M43 Missense_ Hotspot Melanoma Mutation Cutaneous POTEC 18 14542791 SNP C T T Oncomine NM_001137671 p.A119T p.A119 Missense_ Hotspot Melanoma Mutation Clear Cell POTEC 18 14542791 SNP C T T Oncomine NM_001137671 p.A119T p.A119 Missense_ Hotspot Renal Cell Mutation Carcinoma Glioblastoma POTEM 14 20010235 SNP A G G Oncomine NM_001145442 p.V308A p.V308 Missense_ Hotspot Mutation Head and POTEM 14 20010235 SNP A G G Oncomine NM_001145442 p.V308A p.V308 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Cutaneous POTEM 14 20019948 SNP C T T Oncomine NM_001145442 p.M911 p.M91 Missense_ Hotspot Melanoma Mutation Gastric PPIL1 6 36842542 SNP C T T Oncomine NM_016059 p.A3T p.A3 Missense_ Hotspot Adenocarcinoma Mutation Ovarian PPIL1 6 36950519 SNP G G A Oncomine NM_016059 p.A3V p.A3 Missense_ Hotspot Serous Mutation Adenocarcinoma Papillary PPIL1 6 36842542 SNP C T T Oncomine NM_016059 p.A3T p.A3 Missense_ Hotspot Renal Cell Mutation Carcinoma Cutaneous PRAMEF20 1 13743091 SNP C T T Oncomine NM_001099852 p.R94C p.R94 Missense_ Hotspot Melanoma Mutation Glioblastoma PRAMEF20 1 13743092 SNP G A A Oncomine NM_001099852 p.R94H p.R94 Missense_ Hotspot Mutation Melanoma PRB3 12 11420548 SNP C T T Oncomine NM_006249 p.G212E p.G212 Missense_ Hotspot Mutation Cutaneous PRB3 12 11420963 SNP G A A Oncomine NM_006249 p.R74C p.R74 Missense_ Hotspot Melanoma Mutation Head and PRB3 12 11420963 SNP G A A Oncomine NM_006249 p.R74C p.R74 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Cutaneous PRB3 12 11420548 SNP C T T Oncomine NM_006249 p.G212E p.G212 Missense_ Hotspot Melanoma Mutation Cutaneous PRB4 12 11461597 SNP C T T Oncomine NM_002723 p.G107E p.G107 Missense_ Hotspot Melanoma Mutation Cutaneous PRB4 12 11461475 SNP C T T Oncomine NM_002723 p.G148R p.G148 Missense_ Hotspot Melanoma Mutation Cutaneous PRB4 12 11461474 SNP C T T Oncomine NM_002723 p.G148E p.G148 Missense_ Hotspot Melanoma Mutation Cutaneous PROL1 4 71275418 SNP C T T Oncomine NM_021225 p.P125S p.P125 Missense_ Hotspot Melanoma Mutation Cutaneous PROL1 4 71275418 SNP C T T Oncomine NM_021225 p.P125S p.P125 Missense_ Hotspot Melanoma Mutation Cutaneous PROL1 4 71275428 SNP C A A Oncomine NM_021225 p.P128H p.P128 Missense_ Hotspot Melanoma Mutation Cutaneous PROL1 4 71275427 SNP C T T Oncomine NM_021225 p.P128S p.P128 Missense_ Hotspot Melanoma Mutation Cutaneous PRS S37 7 141536973 SNP C T T Oncomine NM_001008270 p.G169E p.G169 Missense_ Hotspot Melanoma Mutation Cutaneous PRSS37 7 141540847 SNP C T T Oncomi NM_001008270 p.M1I p.M1 Missense_ Hotspot Melanoma Mutation Cutaneous PRSS37 7 141536973 SNP C T T Oncomine NM_001008270 p.G169E p.G169 Missense_ Hotspot Melanoma Mutation Cutaneous PRSS37 7 141540847 SNP C T T Oncomine NM_001008270 p.M1I p.M1 Missense_ Hotspot Melanoma Mutation Cutaneous RAB39A 11 107832799 SNP C G G Oncomine NM_017516 p.R119G p.R119 Missense_ Hotspot Melanoma Mutation Colorectal RAB39A 11 107338009 SNP C T T Oncomine NM_017516 p.R119W p.R119 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous RAB39A 11 107832799 SNP C T T Oncomine NM_017516 p.R119W p.R119 Missense_ Hotspot Melanoma Mutation Head and RALB 2 121036297 SNP G A A Oncomine NM_002881 p.M191 p.M19 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Squamous RALB 2 121036296 SNP T C C Oncomine NM_002881 p.M19T p.M19 Missense_ Hotspot Cell Lung Mutation Carcinoma Cutaneous RALB 2 121036296 SNP T A A Oncomine NM_002881 p.M19K p.M19 Missense_ Hotspot Melanoma Mutation Medulloblastoma RANGAP1 22 41652800 SNP A C C Oncomine NM_002883 p.V268G p.V268 Missense_ Hotspot Mutation Cervical RANGAP1 22 41652800 SNP A C C Oncomine NM_002883 p.V268G p.V268 Missense_ Hotspot Squamous Mutation Cell Carcinoma Head and RANGAP1 22 41652800 SNP A C C Oncomine NM_002883 p.V268G p.V268 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Lung RANGAP1 22 41652800 SNP A C C Oncomine NM_002883 p.V268G p.V268 Missense_ Hotspot Adenocarcinoma Mutation Clear Cell RANGAP1 22 41652800 SNP A C C Oncomine NM_002883 p.V268G p.V268 Missense_ Hotspot Renal Cell Mutation Carcinoma Gastric RAP1B 12 69042539 SNP G A A Oncomine NM_015646 p.G12E p.G12 Missense_ Hotspot Adenocarcinoma Mutation Head and RAP1B 12 69042539 SNP G A A Oncomine NM_015646 p.G12E p.G12 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Acute RAP1B 12 67328806 SNP G G A Oncomine NM_015646 p.G12E p.G12 Missense_ Hotspot Myeloid Mutation Leukemia Cutaneous RBMY1D Y 23702641 SNP C T T Oncomine NM_001006120 p.P124L p.P124 Missense_ Hotspot Melanoma Mutation Squamous RBMY1D Y 23702641 SNP C A A Oncomine NM_001006120 p.P124H p.P124 Missense_ Hotspot Cell Lung Mutation Carcinoma Cutaneous RBMY1D Y 23702640 SNP C T T Oncomine NM_001006120 p.P124S p.P124 Missense_ Hotspot Melanoma Mutation Prostate RQCD1 2 219447749 SNP C G G Oncomine NM_005444 p.S87C p.S87 Missense_ Hotspot Adenocarcinoma Mutation Melanoma RQCD1 2 219447749 SNP C G G Oncomine NM_005444 p.S87C p.S87 Missense_ Hotspot Mutation Cutaneous RQCD1 2 219449406 SNP C T T Oncomine NM_005444 p.P131L p.P131 Missense_ Hotspot Melanoma Mutation Cutaneous RQCD1 2 219447748 SNP T C C Oncomine NM_005444 p.S87P p.S87 Missense_ Hotspot Melanoma Mutation Cutaneous S100A7L2 1 153409566 SNP C T T Oncomine NM_001045479 p.G103R p.G103 Missense_ Hotspot Melanoma Mutation Cutaneous S100A7L2 1 153409565 SNP C T T Oncomine NM_001045479 p.G103R p.G103 Missense_ Hotspot Melanoma Mutation Cutaneous S100A7L2 1 153409565 SNP C T T Oncomine NM_001045479 p.G103E p.G103 Missense_ Hotspot Melanoma Mutation Non-Small S100A8 1 153362715 SNP T C C Oncomine NM_002964 p.K49R p.K49 Missense_ Hotspot Cell Lung Mutation Carcinoma, NOS Glioblastoma S100A8 1 153362715 SNP T C C Oncomine NM_002964 p.K49R p.K49 Missense_ Hotspot Head and S100A8 1 153362715 SNP T C C Oncomine NM_002964 p.K49R p.K49 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Thyroid S100A8 1 153362715 SNP T C C Oncomine NM_002964 p.K49R p.K49 Missense_ Hotspot Gland Mutation Papillary Carcinoma Oligodendroglioma SAA2 1 18269491 SNP G A A Oncomine NM_030754 p.S23L p.S23 Missense_ Hotspot Mutation Lung SDR16C5 8 57228627 SNP C A A Oncomine NM_138969 p.A94S p.A94 Missense_ Hotspot Adenocarcinoma Mutation Gastric SDR16C5 8 57228626 SNP G T T Oncomine NM_138969 p.A94D p.A94 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous SDR16C5 8 57228829 SNP C T T Oncomine NM_138969 p.M261 p.M26 Missense_ Hotspot Melanoma Mutation Cutaneous SDR16C5 8 57228854 SNP G A A Oncomine NM_138969 p.S18L p.S18 Missense_ Hotspot Melanoma Mutation Clear Cell SDR16C5 8 57228627 SNP C G G Oncomine NM_138969 p.A94P p.A94 Missense_ Hotspot Renal Cell Mutation Carcinoma Cutaneous SHH 7 155596253 SNP G A A Oncomine NM_000193 p.R244C p.R244 Missense_ Hotspot Melanoma Mutation Lung SHH 7 155596253 SNP G A A Oncomine NM_000193 p.R244C p.R244 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous SHH 7 155596253 SNP G A A Oncomine NM_000193 p.R244C p.R244 Missense_ Hotspot Melanoma Mutation Prostate SLC35G3 17 33520323 SNP C T T Oncomine NM_152462 p.R335K p.R335 Missense_ Hotspot Adenocarcinoma Mutation Infiltrating SLC35G3 17 33520323 SNP C T T Oncomine NM_152462 p.R335K p.R335 Missense_ Hotspot Bladder Mutation Urothelial Carcinoma Glioblastoma SLC35G3 17 33520323 SNP C T T Oncomine NM_152462 p.R335K p.R335 Missense_ Hotspot Mutation Gastric SLC35G3 17 33520323 SNP C T T Oncomine NM_152462 p.R335K p.R335 Missense_ Hotspot Adenocarcinoma Mutation Head and SLC35G3 17 33520392 SNP G C C Oncomine NM_152462 p.A312G p.A312 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Lung SLC35G3 17 33520323 SNP C T T Oncomine NM_152462 p.R335K p.R335 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous SLC35G3 17 33520392 SNP G C C Oncomine NM_152462 p.A312G p.A312 Missense_ Hotspot Melanoma Mutation Cutaneous SLC35G3 17 33520323 SNP C T T Oncomine NM_152462 p.R335K p.R335 Missense_ Hotspot Melanoma Mutation Cutaneous SPATA8 15 97326937 SNP G A A Oncomine NM_173499 p.E18K p.E18 Missense_ Hotspot Melanoma Mutation Head and SPATA8 15 97326937 SNP G A A Oncomine NM_173499 p.E18K p.E18 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Cutaneous SPATA8 15 97326937 SNP G A A Oncomine NM_173499 p.E18K p.E18 Missense_ Hotspot Melanoma Mutation Cervical SPINK13 5 147665577 SNP G A A Oncomine NM_001040129 p.R84H p.R84 Missense_ Hotspot Squamous Mutation Cell Carcinoma Cutaneous SPINK13 5 147665576 SNP C T T Oncomine NM_001040129 p.R84C p.R84 Missense_ Hotspot Melanoma Mutation Cutaneous ST6GAL2 2 107459497 SNP C T T Oncomine NM_032528 p.E313K p.E313 Missense_ Hotspot Melanoma Mutation Colorectal ST6GAL2 2 106816941 SNP G A A Oncomine NM_032528 p.S346L p.S346 Missense_ Hotspot Adenocarcinoma Mutation Endometrial ST6GAL2 2 107460402 SNP C C T Oncomine NM_032528 p.R11Q p.R11 Missense_ Hotspot Endometrioid Mutation Adenocarcinoma Lung ST6GAL2 2 107459730 SNP C A A Oncomine NM_032528 p.G235V p.G235 Missense_ Hotspot Adenocarcinoma Mutation Lung ST6GAL2 2 107460276 SNP G A A Oncomine NM_032528 p.P53L p.P53 Missense_ Hotspot Adenocarcinoma Mutation Squamous ST6GAL2 2 107460166 SNP G A A Oncomine NM_032528 p.H90Y p.H90 Missense_ Hotspot Cell Lung Mutation Carcinoma Squamous ST6GAL2 2 107459731 SNP C A A Oncomine NM_032528 p.G235W p.G235 Missense_ Hotspot Cell Lung Mutation Carcinoma Squamous ST6GAL2 2 107423361 SNP C T T Oncomine NM_032528 p.E455K p.E455 Missense_ Hotspot Cell Lung Mutation Carcinoma Cutaneous ST6GAL2 2 107459497 SNP C T T Oncomine NM_032528 p.E313K p.E313 Missense_ Hotspot Melanoma Mutation Cutaneous ST6GAL 2 107460402 SNP C T T Oncomine NM_032528 p.R11Q p.R11 Missense_ Hotspot Melanoma Mutation Cutaneous ST6GAL2 2 107450509 SNP G A A Oncomine NM_032528 p.S346L p.S346 Missense_ Hotspot Melanoma Mutation Cutaneous ST6GAL2 2 107423361 SNP C T T Oncomine NM_032528 p.E455K p.E455 Missense_ Hotspot Melanoma Mutation Cutaneous ST6GAL2 2 107459496 SNP T A A Oncomine NM_032528 p.E313V p.E313 Missense_ Hotspot Melanoma Mutation Cutaneous ST6GAL2 107459731 SNP C T T Oncomine NM_032528 p.G235R p.G235 Missense_ Hotspot Melanoma Mutation Cutaneous ST6GAL2 2 107460166 SNP G A A Oncomine NM_032528 p.H90Y p.H90 Missense_ Hotspot Melanoma Mutation Cutaneous SYPL1 7 105739611 SNP G A A Oncomine NM_006754 p.P81S p.P81 Missense_ Hotspot Melanoma Mutation Cutaneous SYPL1 7 105739611 SNP G A A Oncomine NM_006754 p.P8S1 p.P81 Missense_ Hotspot Melanoma Mutation Melanoma SYT1 12 79689912 SNP C T T Oncomine NM_005639 p.P180S p.P180 Missense_ Hotspot Mutation Melanoma SYT1 12 79679683 SNP G A A Oncomine NM_005639 p.E95K p.E95 Missense_ Hotspot Mutation Head and SYT1 12 79611355 SNP C T T Oncomine NM_005639 p.A19V p.A19 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Acute SYT1 12 78135485 SNP G G A Oncomine NM_005639 p.A19T p.A19 Missense_ Hotspot Myeloid Mutation Leukemia Cutaneous SYT1 12 79689912 SNP C T T Oncomine NM_005639 p.P180S p.P180 Missense_ Hotspot Melanoma Mutation Cutaneous SYT1 12 79679683 SNP G A A Oncomine NM_005639 p.E95K p.E95 Missense_ Hotspot Melanoma Mutation Prostate SYT1 12 79611355 SNP C T T Oncomine NM_005639 p.A19V p.A19 Missense_ Hotspot Adenocarcinoma Mutation Lung TCEAL8 X 102508844 SNP G T T Oncomine NM_153333 p.R22S p.R22 Missense_ Hotspot Adenocarcinoma Mutation Head and TCEAL8 X 102508843 SNP C T T Oncomine NM_153333 p.R22H p.R22 Missense_ Hotspot Neck Mutation Squamous Clear Cell TCEAL8 X 102508844 SNP G A A Oncomine NM_153333 p.R22C p.R22 Missense_ Hotspot Renal Cell Mutation Carcinoma Prostate TMEM147 19 36037641 SNP C T T Oncomine NM_032635 p.A92V p.A92 Missense_ Hotspot Adenocarcinoma Mutation Glioblastoma TMEM147 19 36037641 SNP C T T Oncomine NM_032635 p.A92V p.A92 Missense_ Hotspot Mutation Cutaneous WFDC5 20 43739300 SNP G A A Oncomine NM_145652 p.R68C p.R68 Missense_ Hotspot Melanoma Mutation Ductal Breast WFDC5 20 43739300 SNP G G A Oncomine NM_145652 p.R68C p.R68 Missense_ Hotspot Carcinoma Mutation Chromophobe WFDC5 20 43739299 SNP C C T Oncomine NM_145652 p.R68H p.R68 Missense_ Hotspot Renal Cell Mutation Carcinoma Clear Cell ZFAND2B 2 220072989 SNP T C C Oncomine NM_138802 p.I149T p.I149 Missense_ Hotspot Renal Cell Mutation Carcinoma Papillary ZFAND2B 2 220072989 SNP T G G Oncomine NM_138802 p.I149S p.I149 Missense_ Hotspot Renal Cell Mutation Carcinoma Non-Small ZNF780A 19 40581109 SNP T C C Oncomine NM_001010880 p.I414V p.I414 Missense_ Hotspot Cell Lung Mutation Carcinoma, NOS Lung ZNF780A 19 40581529 SNP C T T Oncomine NM_001010880 p.V274I p.V274 Missense_ Hotspot Adenocarcinoma Mutation Lung ZNF780A 19 40581535 SNP A C C Oncomine NM_001010880 p.S272A p.S272 Missense_ Hotspot Adenocarcinoma Mutation Oligoastrocytoma ZNF780A 19 40580552 SNP T G G Oncomine NM_001010880 p.Q599H p.Q599 Missense_ Hotspot Mutation Cervical ZNF780A 19 40580552 SNP T G G Oncomine NM_001010880 p.Q599H p.Q599 Missense_ Hotspot Squamous Mutation Cell Carcinoma Gastric ZNF780A 19 40581529 SNP C T T Oncomine NM_001010880 p.V274I p.V274 Missense_ Hotspot Adenocarcinoma Mutation Head and ZNF780A 19 40581109 SNP T C C Oncomine NM_001010880 p.I414V p.I414 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Head and ZNF780A 19 40580552 SNP T G G Oncomine NM_001010880 p.Q599H p.Q599 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Head and ZNF780A 19 40581529 SNP C T T Oncomine NM_001010880 p.V2741 p.V274 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Lung ZNF780A 19 40581109 SNP T C C Oncomine NM_001010880 p.I414V p.I414 Missense_ Hotspot Adenocarcinoma Mutation Squamous ZNF780A 19 40581535 SNP A C C Oncomine NM_001010880 p.S272A p.S272 Missense_ Hotspot Cell Lung Mutation Carcinoma Cutaneous ZNF780A 19 40581535 SNP A C C Oncomine NM_001010880 p.5272A p.5272 Missense_ Hotspot Melanoma Mutation Cutaneous ZNF780A 19 40581109 SNP T C C Oncomine NM_001010880 p.I414V p.I414 Missense_ Hotspot Melanoma Mutation Thyroid ZNF780A 19 40581535 SNP A C C Oncomine NM_001010880 p.S272A p.S272 Missense_ Hotspot Gland Mutation Follicular Carcinoma Thyroid ZNF780A 19 40580552 SNP T G G Oncomine NM_001010880 p.Q599H p.Q599 Missense_ Hotspot Gland Mutation Papillary Carcinoma Lung ZNF844 19 12187394 SNP T C C Oncomine NM_001136501 p.F487L p.F487 Missense_ Hotspot Adenocarcinoma Mutation Glioblastoma ZNF844 19 12187394 SNP T C C Oncomine NM_001136501 p.F487L p.F487 Missense_ Hotspot Mutation Glioblastoma ZNF844 19 12187275 SNP G C C Oncomine NM_001136501 p.R447P p.R447 Missense_ Hotspot Mutation Cervical ZNF844 19 12187394 SNP T C C Oncomine NM_001136501 p.F487L p.F487 Missense_ Hotspot Squamous Mutation Cell Carcinoma Head and ZNF844 19 12187275 SNP G C C Oncomine NM_001136501 p.R447P p.R447 Missense_ Hotspot Neck Mutation Squamous Cell Carcinoma Lung ZNF844 19 12187275 SNP G C C Oncomine NM_001136501 p.R447P p.R447 Missense_ Hotspot Adenocarcinoma Mutation Cutaneous ZNF844 19 12187275 SNP G C C Oncomine NM_001136501 p.R447P p.R447 Missense_ Hotspot Melanoma Mutation Cutaneous ZNF844 19 12187394 SNP T C C Oncomine NM_001136501 p.F487L p.F487 Missense_ Hotspot Melanoma Mutation Oligodendroglioma ZNF845 19 53855196 SNP T C C Oncomine NM_138374 p.M423T p.M423 Missense_ Hotspot Mutation Thyroid ZNF845 19 53855196 SNP T C C Oncomine NM_138374 p.M423T p.M423 Missense_ Hotspot Gland Mutation Papillary Carcinoma Thyroid ZNF845 19 53855197 SNP G A A Oncomine NM_138374 p.M423I p.M423 Missense_ Hotspot Gland Mutation Papillary Carcinoma

The disclosure provides novel gene variants and gene variant-disease state associations. The gene variants can have one or more mutations that result in a variant protein. The gene variants provided herein are associated with certain cancers. The gene variants result in protein variants. The disclosure further provides probes, such as amplification primer sets and detection probes, as well as methods of detection, diagnosis, and treatment and kits that include or detect the gene variants disclosed herein.

The variants are shown as amino acid variants in Tables 7 and 11 with the accession no. or the Entrez nucleotide and/or protein sequence of the parent or wildtype gene provided. The associations with various cancers are shown in Tables 7 and 11. Tables 7 and 11 provide a list of more than 99 genes that were identified using the methods outlined in Example 2. The variations or mutations were not found in the corresponding normal tissue. This is important because in a typical patient, a tumor sample can have 10's-100's of tumor specific variations. However, variations that occur at the same place in multiple patients (and not in the normal tissue) are more significant. 4445 samples (from 4445 patients) were analyzed and list of hotspots was prepared. A number of recurrent mutations were found at the same position in 15-20 different cancer types.

Diagnostics and Kits

Methods of diagnosing, treating, and detecting gene variants and associated disease are contemplated herein. The methods can include detecting gene fusions and/or gene variants in a subject sample. Any number and combination of gene fusions and/or gene variants can be detected in any of the reaction mixtures, compositions, and kits disclosed herein.

In one embodiment, the disclosure provides a composition and a kit comprising a set of probes that specifically recognize the nucleotide sequence that encodes a gene variant selected from Table 7 and/or Table 11. The set of probes can be, for example a set of amplification primers. In another embodiment, provided herein is a composition that includes a set of primers that flank a gene variant that encodes one or more variants in Table 7 and/or Table 11. The reaction mixture of this embodiment can further include a detector probe that binds to a nucleotide sequence including a gene variant selected from Table 7 and/or Table 11. The reaction mixture that includes a detector probe or does not include a detector probe, can further include a polymerase, dNTPs, and/or a uracil DNA deglycosylase (UDG). The polymerase and UDG are typically not from a human origin. The reaction mixture can further include a target nucleic acid, for example a human target nucleic acid. The human target nucleic acid can be, for example, isolated from a biological sample from a person suspected of having a cancer. The cancer can be selected from: BLCA=bladder carcinoma, BRCA=breast carcinoma, CESC=cervical cell carcinoma, COAD=colon adenocarcinoma, GBM=glioblastoma multiforme, HNSC=head and neck squamous cell carcinoma, KIRK=clear cell renal cell carcinoma, KIRP=kidney renal papillary cell carcinoma, LAML=acute myeloid leukemia, LGG=brain lower grade glioma, LIHC=liver hepatocellular carcinoma, LUAD=lung adenocarcinoma, LUSC=squamous cell lung carcinoma, OV=ovarian serous adenocarcinoma, PRAD=prostate adenocarcinoma, READ=rectal adenocarcinoma, SKCM=cutaneous melanoma, STAD=stomach adenocarcinoma, THCA=thyroid carcinoma, and UCEC=uterine corpus endometrioid carcinoma.

In some embodiments a kit is provided, wherein the kit encompasses one or more probes. In some embodiments, the kit encompasses probes for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 150, 200, 250, 500 or more fusion genes. In some embodiments the probe is detectably labeled. In some embodiments the probe hybridizes to the breakpoint present in the gene fusion.

In some embodiments the detection of any one of the gene variants disclosed in Tables 7 and 11 can be combined with the detection of another of the gene variants disclosed in those tables or any of the gene fusions disclosed herein. That is, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 150, 200, 250, 500 or more of the gene variants can be detected in the same reaction. In some embodiments the detected gene variants are those disclosed in Tables 4-6, 7 and 11, 20, and 23 and can be combined with the detection of another of the gene fusion disclosed in those tables. That is, 2, 3, such that 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 150, 200, 250, 500 or more of the gene fusions of can be detected in the same reaction.

The nucleotide sequence that encodes one or more gene variants in Table 7 and/or Table 11 can be any size that encompasses the variation. For example, the nucleotide sequence can be any size that can be easily copied using a primer and/or detected using a probe.

In another embodiment, a set of probes that specifically recognize a nucleic acid coding for a gene variant selected from Table 7 and/or Table 11 (gene variants) is provided. In another embodiment, provided herein is a set of primers that specifically amplify a target nucleic acid that codes for a gene variant selected from Table 7 and/or Table 11. In another embodiment, provided herein is a qPCR assay, such as a TaqMan™ assay or a Molecular Beacons™ assay that specifically amplifies and detects a target nucleic acid that codes for a gene variant selected from Table 7 and/or Table 11.

The disclosure also provides an isolated nucleic acid comprising at least one sequence that includes the variation found in one or more gene variants selected from Table 7 and/or Table 11. The isolated nucleic acid can include a first primer on a 5′ end. Furthermore, the nucleic acid can be single stranded or double stranded.

The disclosure, in other embodiments, provides a kit that includes a detector probe and/or a set of probes, for example, a set of amplification primers that specifically recognize a nucleic acid that codes for a gene variant selected from Table 7 and/or Table 11. For example, in certain embodiments the detector probe or set of amplification primers are designed to amplify and/or detect a nucleic acid that includes at least one of a nucleic acid coding for a gene variant in Table 7 and/or Table 11. The kit can further include, in a separate or in the same vessel, a component from an amplification reaction mixture, such as a polymerase, typically not from human origin, dNTPs, and/or UDG. Furthermore, the kit can include a control nucleic acid. For example the control nucleic acid can include a sequence that includes the gene variant selected from Table 7 and/or Table 11.

A method of detecting a cancer is provided comprising amplifying a nucleic acid that encodes a gene variant selected from Table 7 and/or Table 11, for example the nucleic can include a sequence from one of the accession numbers in Table 7 and/or Table 11 except that the sequence contains the variant that codes for the gene variants in Table 7 and/or Table 11, and detecting the presence of the nucleic acid, wherein the presence of the nucleic acid indicates a cancer is present in the sample. In another method, provided herein is a method of detecting a cancer that includes generating an amplicon that includes a sequence selected from a sequence coding for a gene variant in Table 7 and/or Table 11, and detecting the presence of the nucleic acid, wherein the presence of the nucleic acid indicates bladder, head and neck, or lung squamous cell carcinoma is present in the sample. The amplicon typically includes primers that are extended to form the amplicon. The cancer is selected from bladder carcinoma, breast carcinoma, cervical cell carcinoma, colon adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, clear cell renal cell carcinoma, kidney renal papillary cell carcinoma, acute myeloid leukemia, brain lower grade glioma, liver hepatocellular carcinoma, lung adenocarcinoma, squamous cell lung carcinoma, ovarian serous adenocarcinoma, prostate adenocarcinoma, rectal adenocarcinoma, cutaneous melanoma, stomach adenocarcinoma, thyroid carcinoma, and uterine corpus endometrioid carcinoma.

A kit comprising a set of probes, for example, a set of amplification primers that specifically recognize a nucleic acid comprising a gene variant from Table 7 and/or Table 11 is provided. The kit can further include, in a separate or in the same vessel, a component from an amplification reaction mixture, such as a polymerase, typically not from human origin, dNTPs, and/or UDG. Furthermore, the kit can include a control nucleic acid. For example the control nucleic acid can include a sequence that includes the gene variant from Table 7 and/or Table 11. In certain embodiments, a set of probes that specifically recognize a nucleic acid comprising a gene variant from Table 7 and/or Table 11 is provided.

In another embodiment, a gene variant is provided comprising at least one of the gene variants in Table 7 and/or Table 11.

In another embodiment is a method to detect a cancer selected from bladder carcinoma, breast carcinoma, cervical cell carcinoma, colon adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, clear cell renal cell carcinoma, kidney renal papillary cell carcinoma, acute myeloid leukemia, brain lower grade glioma, liver hepatocellular carcinoma, lung adenocarcinoma, squamous cell lung carcinoma, ovarian serous adenocarcinoma, prostate adenocarcinoma, rectal adenocarcinoma, cutaneous melanoma, stomach adenocarcinoma, thyroid carcinoma, and uterine corpus endometrioid carcinoma in a sample by detecting the presence of a gene variant selected from Table 7 and/or Table 11. Gene variants, for example, can include, but are not limited to ZNF479 variants R110, R295K, R295T, R2951, R3451, R345T, K438T, and T466K (see Table 8).

TABLE 18 Cancer Type Gene Symbol Druggability KM evidence Astrocytoma CXCR2 Y Endometrial Endometrioid Adenocarcinoma CXCR2 Y Squamous Cell Lung Carcinoma CXCR2 Y Cutaneous Melanoma CXCR2 Y Cutaneous Melanoma CXCR2 Y Colorectal Adenocarcinoma IL3 Y Gastric Adenocarcinoma IL3 Y Cutaneous Melanoma KCNK9 Y favorable outcome Endometrial Endometrioid Adenocarcinoma KCNK9 Y Lung Adenocarcinoma KCNK9 Y Squamous Cell Lung Carcinoma KCNK9 Y poor outcome Non-Small Cell Lung Carcinoma, NOS S100A8 Y Glioblastoma S100A8 Y Head and Neck Squamous Cell Carcinoma S100A8 Y Thyroid Gland Papillary Carcinoma S100A8 Y Cutaneous Melanoma SHH Y Lung Adenocarcinoma SHH Y Cutaneous Melanoma CCDC61 poor outcome Cutaneous Melanoma CCDC61 poor outcome Cutaneous Melanoma CNTN5 poor outcome Cutaneous Melanoma CNTN5 poor outcome Cutaneous Melanoma CNTN5 poor outcome Cutaneous Melanoma CNTN5 poor outcome Colorectal Adenocarcinoma CNTN5 poor outcome Colorectal Adenocarcinoma CNTN5 poor outcome Cutaneous Melanoma CNTN5 poor outcome Cutaneous Melanoma CNTN5 poor outcome Cutaneous Melanoma CNTN5 poor outcome Cutaneous Melanoma EDDM3A poor outcome Cutaneous Melanoma FABP1 poor outcome Lung Adenocarcinoma OR2L13 poor outcome Cutaneous Melanoma OR4E2 poor outcome Cutaneous Melanoma OR4E2 poor outcome Cutaneous Melanoma OR4E2 poor outcome Cutaneous Melanoma PRSS37 poor outcome Cutaneous Melanoma PRSS37 poor outcome Cutaneous Melanoma SPINK13 poor outcome Endometrial Endometrioid Adenocarcinoma ST6GAL2 poor outcome

Table 18 provides druggablility or prognostic associations that were filtered from Table 11. Table 18 provides the cancer type, gene symbol, druggability (Y=yes), and KM evidence for the genes identified in Table 11 as druggable. The KM Evidence column provides the Kaplan-Meier evidence. The KM evidence indicates if the event type supports good or poor prognosis in the particular cancer type.

Targeted Treatment

In at least one embodiment, the gene fusions and/or gene variants can be used to identify targeted therapies. Targeted therapies can include the identification of agents that specifically interact with the gene fusion and/or gene variant. Targeted therapies can include, but are not limited to, antibody therapies, antisense therapies and small molecule therapies. Antisense therapies are discussed in more detail under the heading “antisense.”

Compositions and methods for inactivating nucleic acid molecules involve, in part, the use of molecules with nucleic acid regions with sequence complementarity to the nucleic acid molecule which is the subject of desired inactivation (i.e., a target nucleic acid molecule). Methods of the invention can be used for inactivation of gene fusions and/or gene variants associated with specific cancers. Thus, antisense molecules can be identified that are complementary to any of the gene fusions or gene variants identified herein.

Small molecules are low molecular weight (<800 Daltons) organic compounds that may serve as enzyme substrates or regulators of biological processes, with a size on the order of 10⁻⁹ m. In pharmacology, the term is usually used for a molecule that binds to a protein or nucleic acid, and acts as an effector, altering the activity or function of the protein or nucleic acid. Small molecules can be tested for effector functions by expressing a gene fusion or variant in a cellular assay and identifying small molecules that inhibit expression or activity of the gene fusion or variant.

Druggability is a term used in drug discovery to describe a biological target such as a protein that is known to bind or is predicted to bind with high affinity to a drug. Furthermore, the binding of the drug to a druggable target alters the function of the target with a therapeutic benefit to the patient. The term “drug” herein includes small molecules (low molecular weight organic substances) but also has been extended to include biologic medical products such as therapeutic monoclonal antibodies. In at least one embodiment, the gene fusion or gene variant can be used to identify a druggable target. Table 8 provides a list of druggable targets that have been identified from Tables 1-3 and 7. For example, the TPM1/ALK gene fusion is a druggable target because, as shown in Table 8, diseases for which ALK is involved can be treated with crizotinib. Thus, if a gene fusion includes ALK, the cancer may be treatable with crizotinib. Further if a gene variant includes a mutation in ALK, the cancer may be treatable with crizotinib.

Similarly, Table 21 provides a list of druggable targets that have been identified from Table 19 and Table 24 a list of druggable targets that have been identified from Table 22.

TABLE 8 Druggable genes from Table 1 Pre- registration Druggable (pre- Gene Approved approval) Phase III Phase II Phase I Preclinical ALK crizotinib N N AP-26113; X-396; ASP- NMS-E628; RG-7853; 3026 aurora kinase + LDK-378; ALK inhibitor TSR-011 (Sareum, AstraZeneca); ALK inhibitors (AstraZeneca, Cephalon, Aurigene); ARN- 5032; DLX-521 CASR cincacalcet N N N N N hydrochloride EGFR erlotinib; Afatinib zalutumumab; BMS-690514; marizomib; STP-503; SN- panitumumab; neratinib; varlitinib; AC- CUDC-101; 29966; MT-062; cetuximab; dovitinib 480; AZD- MM-151; STP-801 nepidermin; lactate; XL- 8931; Sym- AL-6802; S- gefitinib; 647; 004; 222611; nimotuzumab; rindopepimut; imgatuzumab; ABT-806; vandetanib; necitumumab; AVL-301; antroquinonol; lapatinib dacomitinib AVL-301; GT-MAB ditosylate; poziotinib; 5.2-GEX; icotinib MEHD- epitinib; hydrochloride; 7945A; PR- theliatinib; 610; cipatinib; AMG-595 FGFR3 ponatinib Masitinib dovitinib ENMD-2076; JNJ- N lactate AZD-4547 42756493; BGJ-398; LY-2874455; S-49076 GNAS N N N N N N JAK2 ruxolitinib N SAR-302503; AT-9283; AC-430; SB- ON-044580; (for pacritinib momelotinib; 1317 INCB-16562; idiopathic gandotinib; NVP-BSK805; myelofibrosis) BMS-911543; TP-0413; MRLB- NS-018 11055; CPL-407- 22 NOTCH1 N N N N OMP-52M51 Debio-0826; TR- 4; Notch antibody (AVEO); Notch1 inhibitors (Interprotein) NTRK1 N N N milciclib N tyrosine kinase maleate inhibitors (Bristol-Myers Squibb); PLX- 7486 PIK3CA N N perifosine; ZSTK-474; INK-1117; LOR-220; AEZS- buparlisib; PX-866; GSK- 129; SB-2343; pictilisib; XL- 2126458; WX-037; 765; XL-147; CUDC-907; PI3/Mnk kinase BEZ-235; GDC-0032; inhibitors PKI-587; PF- PWT-33597; (Progenics); 04691502; PF- DS-7423; AEZS-132; CLR- 04691502; GDC-0084; 1401; PI3/mTOR BAY-80-6946; BAY- kinase inhibitors BYL-719; 1082439; PI3 (Amgen); AEZS- kinase/mTOR 136; HM-032; inhibitor AMG-511; (Lilly) anticancer therapy (Sphaera Pharma); HMPL- 518; GNE-317; mTOR inhibitor/PI3 kinase inhibitor (Lilly); CUDC908; PF- 06465603; AEZS- 134; RET sorafenib; N motesanib N MG-516; RET vandetanib; diphosphate; kinase inhibitor; sunitinib SAR-302503; NMS-173 malate; apatinib cabozantinib; regorafenib ROS1 crizotinib N N N N N ALK crizotinib N N AP-26113; X-396; ASP- NMS-E628; RG-7853; 3026 aurora kinase + LDK-378; ALK inhibitor TSR-011; (Sareum, NMS-E628 AstraZeneca); ALK inhibitors (AstraZeneca, Cephalon, Aurigene); ARN- 5032; DLX-521 NTRK1 N N N milciclib N tyrosine kinase maleate inhibitors (Bristol-Myers Squibb); PLX- 7486 VIM N N N pritumumab N N PTK2 PF-04554878 GSK- CFAK-C4; FAK 2256098; BI- inhibitors 853520; VS- (Varastem, 4718 Takeda); CTX- 0294945; CTX- 0294945 BRS3 N N N N N N TP53 Gendicine N N quinacrine; RG-7388; PXN-527; APR-246; SGT-53; ORCA-010; TR- ISA-102 CBLC-137; 2; ALT-802; SAR-405838 OBP-702 STAT3 N N N brivudine; OPB-51602 CLT-005; GLG- OPB-31121; 101; GLG-202; anatabine GLG-302; GLG- citrate; ISIS- 401; PNT-500 STAT3Rx NOTCH2 N N N OMP-59R5 N N MET cabozantinib; N tivantinib; MGCD-265; AMG-208; X-379; metatinib; crizotinib rilotumumab; foretinib; TAS-115; PRS-110; ASP- onartuzumab; ficlatuzumab; volitinib; 08001; ARGX- BMS-777607; SAR-125844; 111; DCC-2701; golvatinib; S-49076 DCC-2721; MG- INCB-028060; 516; AL-2846; LY-2875358 CG-206481; T- 1840383; cMet- EGFR dual inhibitors (CrystalGenomics); bispecific antibodies (Hoffmann-La Roche) CDH1 N N N N N N TOP1 belotecan N cositecan; gimatecan; irinotecan, camptothecin hydrochloride; irinotecan, camptothecin, liposomal, (Aphios); irinotecan HyACT; Calando; Yakult; HM- irinotecan hydrochloride; irinotecan, irinotecan HCl + 30181A; (BioAlliance); topotecan PharmaEngine; floxuridine, namitecan; cisplatin + etirinotecan Celator; camptothecin irinotecan pegol firtecan pegol; prodrug, (Celator); APH- TLC-388 Mersana; 0804; irinotecan hydrochloride; labetuzumab- (Champions); hRS7-SN-38; SN-38; Genz- SER-203; SN-38; irinotecan 644282; topotecan + bead, simmitecan vincristine Biocompatibles hydrochloride (LipoCure); prodrug topotecan (EnduRx Pharmaceuticals) RARA tamibarotene N N IRX-5183 N N ERBB2 trastuzumab; trastuzumab, neratinib; XL- lapuleucel-T; Her-VAXX; Lovaxin B; TH-1 trastuzumab Enhanze 647; AVX-901; VM-206; (Algeta); emtansine; dacomitinib; AE-37; BMS- ARRY-380; trastuzumab- pertuzumab; nelipepimut-S; 690514; JNJ- antibody lapatinib trastuzumab MVA-BN- 26483327; S- conjugates ditosylate; (Celltrion, HER2; 222611; (Synthon); catumaxomab; Biocad, varlitinib; doxorubicin CUDC-101; Her- afatinib Biocon, MM-111; AC- (Merrimack); 2/neu Stradobody Synthon, 480; ovarian cipatinib; (Gliknik); ARX- Harvest Moon, cancer vaccine TrasGEX; 788; Etbx-021; Aryogen) (Generex); trastuzumab SN-34003; IBI- margetuximab; (Hanwha 302; NT-004; poziotinib; PR- Chemical); ICT-140; ONS- 610 trastuzumab 1050; Sym-013; (Pfizer); IDN- anti-HER2 X anti- 6439 CD3 (Emergent Biosolutions); Z- 650; breast cancer vaccine (Cel-Sci); JNJ-28871063; trastuzumab (PlantForm, BioXpress, biOasis Technologies, Stada, Natco, Curaxys, Oncobiologics, Alteogen, Mabion) ALK crizotinib N N AP-26113; X-396; ASP- NMS-E628; RG-7853; 3026 aurora kinase + LDK-378; ALK inhibitor TSR-011; (Sareum, NMS-E628 AstraZeneca); ALK inhibitors (AstraZeneca, Cephalon, Aurigene); ARN- 5032; DLX-521 NTRK1 N N N milciclib N tyrosine kinase maleate inhibitors (Bristol-Myers Squibb); PLX- 7486 LTK crizotinib N N N N N BRAF pazopanib; N N RAF-265; XL- ARQ-761; AB-024; b-raf vemurafenib; 281; LGX-818 ARQ-736 inhibitors dabrafenib (Sareum); BRAF kinase inhibitor (Selexagen Therapeutics); BeiGene-283; DP-4978; TL-241

Table 8 provides a list of 11 druggable targets that were identified in the gene fusions in Tables 1-3 or gene variants in Tables 7 and 11. Tables 16 and 17 provide an analysis of other druggable targets within Tables 1-3 or gene variants in Tables 7 and 11. Tables 8, 16 and 17 provide information about druggable targets including the gene name whether the drug has been approved (N=no) by the U.S. Food and Drug Administration (FDA), if the drug has not been approved, which phase the clinical trial is in (Pre-registration, Phase III, Phase II, Phase I, and preclinical). For example, the drug associated with the NOTCH1 gene has not been approved, but is in Phase 1 of clinical trials (see OMP-52M51) as of this writing.

Approved drugs include, but are not limited to, crizotinib for diseases having ALK gene fusions and cincacalcet hydrochloride for diseases having CASR gene fusions. A number of approved drugs have been identified for gene fusions having EGFR, including, but not limited to, erlotinib; panitumumab; cetuximab; nepidermin; gefitinib; nimotuzumab; vandetanib; lapatinib ditosylate; and icotinib hydrochloride. The approved drug ponatinib has been identified for diseases having FGFR3, ruxolitinib has been identified for diseases having JAK2 gene fusions. A number of approved drugs have been identified for gene fusions having RET, including but not limited to, sorafenib; vandetanib; sunitinib malate; cabozantinib; and regorafenib. The approved drug crizotinib has been identified for diseases having ROS1. Additional drugs that may prove useful include, but are not limited to, zrizotinib, afatinib, masitinib, zalutumumab, neratinib, dovitinib lactate, XL647, rindopepimut, nectumumab, dacomitinib, SAR-302503, pacritinib, perifosine, buparlisib, motesinib diphosphate, and apatinib.

Methods provided herein can include delivering a drug to a subject or a patient. The drug can be an approved drug according to a governmental drug regulatory authority, such as the FDA, or the drug can be in any of the stages before the approved stage. In illustrative aspects, the drug is an FDA-approved drug. In other aspects the drug can be in a pre-clinical, Phase I, Phase II, Phase III, or pre-approval stage. In certain aspects, the methods provided herein include delivering one or more than one of the drugs listed in Tables 8, 16 and 17 to a subject. Where genetic events are identified in a subject that involve more than one gene listed in Tables 8, 16 and 17, methods provided herein can include delivering more than one drug, particularly delivering drugs associated with the different genes affected by the identified genetic events.

Antisense

Antisense technology has been applied to inhibit the expression of various oncogenes. For example, Craf-1 cDNA fragments in an antisense orientation, brought under the control of an adenovirus 2 late promoter introduced into a human squamous carcinoma resulted in a greatly reduced tumorigenic potential relative to cells transfected with control sense transfectants. Similarly, a Cmyc antisense construct accelerated differentiation and inhibited G₁ progression in Friend Murine Erythroleukemia cells. Antisense methodology takes advantage of the fact that nucleic acids tend to pair with “complementary” sequences. Complementary sequences are those polynucleotides which are capable of base-pairing according to the standard Watson-Crick complementarity rules. Targeting double-stranded (ds) DNA with polynucleotides leads to triple-helix formation; targeting RNA will lead to double-helix formation. Antisense polynucleotides, when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability. Antisense RNA constructs, or DNA encoding such antisense RNA's, may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.

Antisense can be under transcriptional control of a promoter. A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.

In certain instances, an antisense expression construct will comprise a virus or engineered construct derived from a viral genome. Where a cDNA insert is employed, a polyadenylation signal to effect proper polyadenylation of the gene transcript may be included. The nature of the polyadenylation signal is not believed to be crucial and any such sequence may be employed. A terminator can be used to enhance message levels and to minimize read through from the cassette into other sequences.

Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene fusion or gene variant disclosed herein. The most effective antisense constructs include regions complementary to intron/exon splice junctions. One embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary, depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.

The word “complementary” with respect to antisense means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.

In vivo, ex vivo or in vitro delivery of antisense can involve the use of vectors. One effective vector for antisense delivery is an adenovirus expression vector. “Adenovirus expression vector” is meant to include those constructs containing adenovirus sequences sufficient to support packaging of the construct and to express an antisense polynucleotide that has been cloned therein. The expression vector can include a genetically engineered form of adenovirus. Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target-cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging.

Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 10⁹-10¹¹ plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus, demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.

The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription. The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5′ and 3′ ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome.

In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed. When a recombinant plasmid containing a human cDNA, together with the retroviral LTR and packaging sequences is introduced into a cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media. The recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells.

A novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification could permit the specific infection of hepatocytes via sialoglycoprotein receptors.

Other viral vectors may be employed as expression vectors. Vectors derived from viruses such as vaccinia virus, adeno-associated virus (AAV) and herpes viruses may be employed.

In order to effect expression of sense or antisense gene constructs, the expression vector may be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. As described above, one mechanism for delivery is via viral infection where the expression construct is encapsidated in an infectious viral particle.

Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated. These include calcium phosphate precipitation DEAE-dextran, electroporation, direct microinjection, DNA-loaded liposomes, and lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection. Some of these techniques may be successfully adapted for in vivo or ex vivo use.

Pharmaceutical Compositions—Where clinical applications are contemplated, pharmaceutical compositions can be produced—either gene delivery vectors or engineered cells—in a form appropriate for the intended application. Generally, this entails preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.

Appropriate salts and buffers are used to render delivery vectors stable and allow for uptake by target cells. Buffers also will be employed when recombinant cells are introduced into a patient. Aqueous compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase “pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated.

The expression vectors and delivery vehicles may be administered via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions.

An effective amount of the therapeutic agent is determined based on the intended goal. The term “unit dose” refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired response in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the subject to be treated, the state of the subject and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual.

Therapeutic Kits—All the essential materials and reagents required for inhibiting tumor cell proliferation may be assembled together in a kit. This generally will comprise selected expression vectors, viruses or cells. Also included may be various media for replication of the expression vectors and host cells for such replication. Such kits will comprise distinct containers for each individual reagent. The kits may also include an instruction sheet defining (i) administration of the antisense expression vector construct; (ii) the antisense expressing viruses; and (iii) the antisense expressing cells.

In some embodiments, an interfering (iRNA or siRNA) is provided. In some embodiments the iRNA is complementary to the breakpoint of a fusion gene.

Methods Associated with Clinical Outcome Discoveries

Tables 15 and 39 provided herein, contain more than 100 genetic events, including gain-of-function mutations, loss-of-function mutations, in-peak gene amplification/deletions, and fusion events for various cancer types that are associated with a clinical outcome with high statistical significance (q<0.1). Accordingly, provided herein are methods for delivering a treatment to a subject, methods for determining whether a subject receives a treatment, methods for determining whether to deliver a treatment, and methods for delivering a report. The treatment, in certain illustrative embodiments, is a drug. As non-limiting examples, the drug can be a drug listed in Tables 8, 16 and 17, especially where the method involves a genetic event that affects the gene listed for the drug in Tables 8, 16 and 17. In other examples, the drug can be any drug approved by a regulatory agency, or any drug in a stage of development before approval, as discussed herein.

Accordingly, in another embodiment, a method of delivering a treatment to a subject is provided, wherein the method includes detecting a genetic event identified in Table 15, and treating the subject, wherein the treatment is believed to positively affect the clinical outcome of cancer patients having the genetic event and/or is believed to affect a biological pathway associated with the genetic event. This embodiment can be considered a method for determining if a subject receives a treatment or a method for determining whether to deliver or perform a treatment to or on a subject. Thus, provided herein is a method for determining if a subject receives a drug, the method includes detecting a genetic event identified in Table 15 and/or 39, and then delivering a drug to the subject if the detected genetic event is listed in Table 15 and/or 39, wherein the drug is believed to positively affect the clinical outcome of patients having the genetic event. In illustrative aspects of these embodiments, the genetic event is associated with a gene found in Tables 8, 16 and 17, and the drug is listed in Tables 8, 16 and 17, as a companion for that gene. The subject is typically a subject that has a cancer of the type listed in Table 15 and/or 39. In illustrative aspects of this embodiment the genetic event is associated with a poor prognosis for the subject, who is afflicted with a cancer, typically the cancer listed in Table 15 and/or 39 for which the poor prognosis is associated with that genetic event.

In another embodiment, provided herein is a method of delivering a report, wherein the method includes detecting a genetic event identified in Table 15 and/or 39 and delivering to a medical professional, a report that provides a predicted clinical outcome associated with that genetic event for a cancer of the subject. The medical professional can be, as non-limiting examples, a physician, genetic counselor, or other medical professional. Typically, the physician, genetic counselor, or other medical professional have a professional relationship with the subject, such as a patient/doctor relationship. The report can be a paper report or can be an electronic report delivered to the medical professional over a computer network. The method and report can include one or more of the genetic events and associated prognosis identified in Table 15 and/or 39.

In another embodiment, provided herein is a method for determining which treatment to administer to a subject, the method includes detecting a genetic event listed in Table 15, and administering the treatment depending on the genetic event that is detected. In illustrative embodiments, the treatment is an aggressive treatment, such as a treatment that will involve more pain and suffering for the patient as a result of the treatment, if the detected genetic event is associated with a poor prognosis. In related embodiments the treatment is a more aggressive treatment if the detected genetic event is associated with a poor prognosis and a less aggressive treatment if the detected genetic event is another genetic event, especially if the detected genetic event is identified in Table 15 and/or 39 as indicating a good prognosis. For example, if a AADAC gene deletion, an amplification of the CHD1L gene, the FMO5 gene, or the PRKAB2 gene, or a combination thereof, is detected in a lung cancer adenocarcinoma patient, the patient may be treated with an aggressive chemotherapeutic drug regimen. If these genetic events are not detected in the patient, then the patient may be monitored but the chemotherapeutic drug may not be administered.

In another embodiment, provided herein is a method for determining whether to treat a cancer patient, the method includes detecting a genetic event listed in Table 15 and/or 39, and treating the subject if a genetic event is detected that is associated in Table 15 with a poor prognosis. In another embodiment, provided herein is a method for determining whether to treat a cancer patient, the method includes detecting a genetic event listed in Table 15 and/or 39, and not treating the subject if a genetic event is detected that is associated in Table 15 and/or 23 with a good prognosis. In another embodiment, provided herein is a method for determining whether to treat or monitor a cancer patient, the method includes detecting a genetic event listed in Table 15 and/or 39, and monitoring, but not treating the subject if a genetic event is detected that is associated in Table 15 and/or 39 with a good prognosis. Treatment may be administered at a later time if the monitoring detects recurrence or progression of the cancer.

In certain aspects of these embodiments of the invention that relate to methods provided herein based on the clinical outcomes associated with genetic events in Table 15 and/or 39, for example methods for delivering a treatment to a subject or determining whether to deliver a treatment to a subject, or determining which treatment to administer or deliver, or methods for delivering a report, the subject can be identified as having any of the types of genetic events and any of the specific genetic events listed in Table 15 and/or 39. For example, the genetic event can be a gain-of-function mutation, loss-of-function mutation, a gene amplification or deletion, typically an in-peak gene amplification/deletion, or a fusion event. In certain illustrative embodiments the genetic event is identified in Table 15 and/or 39 of having a q-value of 1×10⁻³ or less, 1×10⁻⁴ or less, or 1×10⁻⁵ or less. In certain aspects, the genetic event is listed in Table 15 and/or 39 as involving a druggable gene. For example, the genetic event can be a genetic event listed in Table 15 and/or 39 associated with a gene that is a preclinical drug target. As a non-limiting example, provided herein is a method for determining which treatment or course of treatment to administer to a patient who has ovarian cancer, for example ovarian serous cystadenocarcinoma, wherein the method includes detecting or otherwise determining an amplification of the ID1 or BCL2L1 gene and administering the treatment. The treatment in illustrative embodiments, is an approved treatment for BCL2L1, such as a currently FDA-approved BCL2L1 treatment, wherein a BCL2L1 amplification is detected.

Methods are known to skilled artisans for detecting the types of genetic events listed in Table 15 and/or 39. Those methods can include nucleic acid sequencing methods or amplification methods, such as PCR or isothermal amplification methods, or combinations thereof. Those methods can include providing a primer that is designed to bind to a gene identified in Table 15 and/or 39 or bind upstream of a gene identified in Table 15 and/or 39. Thus, provided herein are reaction mixtures and kits that include a nucleic acid sample for a subject and one or more primers that bind to, or upstream from, a gene identified in Table 15 and/or 39. Typically, the gene is associated with a genetic event in Table 15 and/or 39, and the subject has a cancer identified in Table 15 and/or 39 as having a prognosis associated with the genetic event. The kit can also include a control nucleic acid that is bound by the primer as disclosed herein for various embodiments of the invention. The reaction mixture can also include a polymerase as disclosed herein for various embodiments of the invention.

In certain aspects of these embodiments of the invention that relate to methods provided herein based on the clinical outcomes associated with genetic events in Table 15 and/or 39, for example methods for delivering a treatment to a subject or determining whether to deliver a treatment to a subject, methods for determining which treatment to deliver, or methods for delivering a report to a medical professional, the genetic event can include more than one of the genetic events identified in Table 15 and/or 39. In certain aspects, a method according to this embodiment detects 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the genetic events identified in Table 15, especially those identified with the same prognosis for a given cancer type. For example, the method can include detecting a genetic event in a breast cancer patient and administering a treatment to the patient, where the detected genetic event includes a gene amplification of two or more of the BRF2, ERLIN2, GPR124, PROSC, and TAB11FI genes. In another example, the method includes detecting two or more genetic events in a subject afflicted with a lower grade glioma and administering a treatment to the subject, wherein the genetic event is at least two of an amplification of the EGFR or SEC61G gene, an amplification of the CDK4, CYP27B1, MARCH9, TSPAN31, or AGAP2 gene, a gain of function mutation in the EGFR gene, or a deletion of the CDKN2A, CDKN2B, or MTAP gene. In another aspect, the method includes detecting a genetic event associated with a poor prognosis and the genetic event is identified in Tables 8, 16, 17, Table 15 and/or 39 as being a target for a current drug in pre-clinical trials or an approved drug, such as an FDA approved drug.

In certain aspects of these embodiments of the invention that relate to methods provided herein based on the clinical outcomes associated with genetic events in Table 15 and/or 39, for example methods for delivering a treatment to a subject or determining whether to deliver a treatment to a subject, or determining which treatment to administer or deliver, or methods for delivering a report, the genetic event can be a specific genetic event identified in one of the other tables herein. A skilled artisan can identify which general type of genetic event in Table 15 and/or 39 a specific genetic event in one of the other tables will fall under.

Computer Implemented Systems

Computer systems can be utilized to in certain embodiments of the disclosure. In various embodiments, computer system can include a bus or other communication mechanism for communicating information, and a processor coupled with bus for processing information. In various embodiments, computer system 100 can also include a memory, which can be a random access memory (RAM) or other dynamic storage device, coupled to bus for determining base calls, and instructions to be executed by processor. Memory also can be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor. In various embodiments, computer system can further include a read only memory (ROM) or other static storage device coupled to bus for storing static information and instructions for processor. A storage device, such as a magnetic disk or optical disk, can be provided and coupled to bus for storing information and instructions.

In various embodiments, computer system can be coupled via bus to a display, such as a cathode ray tube (CRT) or liquid crystal display (LCD), for displaying information to a computer user. An input device, including alphanumeric and other keys, can be coupled to bus for communicating information and command selections to processor. Another type of user input device is a cursor control, such as a mouse, a trackball or cursor direction keys for communicating direction information and command selections to processor and for controlling cursor movement on display. This input device typically has two degrees of freedom in two axes, a first axis (i.e., x) and a second axis (i.e., y), that allows the device to specify positions in a plane.

A computer system can perform the present teachings. Consistent with certain implementations of the present teachings, results can be provided by computer system 100 in response to processor executing one or more sequences of one or more instructions contained in memory. Such instructions can be read into memory from another computer-readable medium, such as storage device. Execution of the sequences of instructions contained in memory can cause processor to perform the processes described herein. Alternatively hard-wired circuitry can be used in place of or in combination with software instructions to implement the present teachings. Thus implementations of the present teachings are not limited to any specific combination of hardware circuitry and software.

In various embodiments, the term “computer-readable medium” as used herein refers to any media that participates in providing instructions to processor for execution. Such a medium can take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Examples of non-volatile media can include, but are not limited to, optical or magnetic disks, such as storage device. Examples of volatile media can include, but are not limited to, dynamic memory, such as memory. Examples of transmission media can include, but are not limited to, coaxial cables, copper wire, and fiber optics, including the wires that comprise bus.

Common forms of non-transitory computer-readable media include, for example, a floppy disk, a flexible disk, hard disk, magnetic tape, or any other magnetic medium, a CD-ROM, any other optical medium, punch cards, paper tape, any other physical medium with patterns of holes, a RAM, PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.

In accordance with various embodiments, instructions configured to be executed by a processor to perform a method are stored on a computer-readable medium. The computer-readable medium can be a device that stores digital information. For example, a computer-readable medium includes a compact disc read-only memory (CD-ROM) as is known in the art for storing software. The computer-readable medium is accessed by a processor suitable for executing instructions configured to be executed.

In accordance with the teachings and principles embodied in this application, methods, systems, and computer readable media that can efficiently collect, analyze, store, transfer, retrieve, and/or distribute information across multiple sites and/or entities, including genomic and/or patient information, are provided.

In one embodiment, a system is provided for determining whether one or more gene fusion and/or variant is present in a sample. The system can further determine identify a disease state, such as cancer, associated with the one or more gene fusion and/or gene variant, as well as an appropriate treatment in accordance with the mutation status. In certain embodiments, the system comprises a processor in communication with a sequencing instrument that receives sequencing data.

In some embodiments, the processor can execute one or more variant calls. In some embodiments, the processor can provide, filter, and/or annotate predictions.

EXAMPLES

In the following examples, methods were used to identify gene fusions and gene variants associated with a panel of 19 cancers in 4,225 cancer patient samples. The gene fusions and gene variants are then used to produce diagnostic methods to identify a predisposition for cancer, to diagnose cancer, to stage cancer, to provide a prognosis and to identify a druggable cancer. Methods are provided to provide targeted therapy for the cancer based on the identification of gene fusions.

Example 1: High-Throughput Systematic Analysis of Paired-End Next-Generation Sequencing Data to Characterize the Gene Fusion Landscape in Cancer

4,225 cancer patient samples across 19 diseases were processed with deFuse McPherson et al. “deFuse: an algorithm for gene fusion discovery in tumor RNASeq data” PLoS Comp. Bio. 2011. and TopHat (Kim et al. “TopHat-Fusion: an algorithm for discovery of novel fusion transcripts” Genome Biology 2011) gene fusion calling software using a cloud-based computation infrastructure. Filtering criteria were identified for gene fusion events that enriched for high confidence, chemically validated gene fusion events.

Gene fusions encode oncogenic drivers in hematologial and solid tumors and are often associated with dramatic clinical responses with the appropriate targeted agents. Massively parallel paired-end sequencing can identify structural rearrangements in tumor genomes and transcriptomes. However, computational methods to identify gene fusions are varied, still evolving and largely trained on cell line data. Systematic methods were developed to characterize known oncogenic gene fusions and to discover novel gene fusions in cancer. RNASeq data for approximately 3,400 clinical cases from 16 cancer types was obtained from the Cancer Genomics Hub (CGHub) of the Cancer Genome Atlas (TCGA). The performance of several gene fusion callers was surveyed and two were chosen (deFuse and TopHat) for further method development with the goal of supporting both single and paired end data. An analysis pipeline was developed and executed in parallel on a high-performance computing cluster. Filtering and annotation was conducted on aggregated data as a post-processing step to enable exploratory analyses of various filters. Filtering approaches were optimized on datasets that included known standards (e.g., TMPRSS2.ERG in prostate adenocarcinoma, PML.RARA in acute myeloid leukemia, etc.) to enrich for these and other gene fusions with correct 5′-3′ orientation while excluding cases with ambiguous breakpoints and spanning reads, alignment errors, and read through transcripts from adjacent genes. Predicted fusions were summarized based on the occurrence of unique genes participating in fusion with multiple partners and of unique gene pairs, each within specific diseases. Elevated expression was observed after the predicted breakpoint of the 3′ gene in cases positive for predicted fusions and added important confirmatory evidence. Pan-disease fusions and multi-partner fusion events broadened the clinical population scope of gene fusion events.

All single-end data was processed using TopHat and all paired-end data was processed using deFuse. TopHat has been shown to be effective with longer 75 bp single-end data. The deFuse algorithm is not compatible with single-end data and has been designed to leverage read pairs. The pre-processing data and Detect fusions: deFuse TopHat steps were executed in parallel for all samples on a high-performance computing cluster. The filtering and annotation was conducted on the aggregated data as a post-processing step to enable filtering criteria to minimize false positive fusions. The list of priority fusions was validated with RNASeq Exon Expression data.

TCGA Data Source: All RNASeq data for gene fusion analysis was obtained from the Cancer Genomics Hub (CGHub), the current repository for TCGA genomic data—cqhub.ucsc.edu. Table 9 lists the TCGA sample counts downloaded and processed for M2 and M3:

TABLE 9 TCGA samples processed Cancer Type Cancer Type Abbreviation Samples Center Instrument Bladder Urothelial Carcinoma BLCA 122 UNC-LCCC Illumina HiSeq 2000 Breast invasive carcinoma BRCA 841 UNC-LCCC Illumina HiSeq 2000 Cervical squamous cell carcinoma and CESC 88 UNC-LCCC Illumina HiSeq 2000 endocervical adenocarcinoma Colon adenocarcinoma COAD* 196 UNC-LCCC Illumina GA IIx Glioblastoma multiforme GBM 167 UNC-LCCC Illumina HiSeq 2000 Head and Neck squamous cell HNSC 302 UNC-LCCC Illumina HiSeq 2000 carcinoma Kidney Chromophobe KICH 66 UNC-LCCC Illumina HiSeq 2000 Kidney renal clear cell carcinoma KIRC 480 UNC-LCCC Illumina HiSeq 2000 Kidney renal papillary cell carcinoma KIRP 76 UNC-LCCC Illumina HiSeq 2000 Acute Myeloid Leukemia LAML 179 BCCAGSC Illumina GA IIx Brain Lower Grade Glioma LGG 184 UNC-LCCC Illumina HiSeq 2000 Liver hepatocellular carcinoma LIHC 34 UNC-LCCC Illumina HiSeq 2000 Lung adenocarcinoma LUAD 345 UNC-LCCC Illumina HiSeq 2000 Lung squamous cell carcinoma LUSC 221 UNC-LCCC Illumina HiSeq 2000 Ovarian serous cystadenocarcinoma OV 417 BCCAGSC Illumina HiSeq 2000 Pancreatic adenocarcinoma PAAD 31 UNC-LCCC Illumina HiSeq 2000 Prostate adenocarcinoma PRAD 140 UNC-LCCC Illumina HiSeq 2000 Rectum adenocarcinoma READ* 71 UNC-LCCC Illumina GA IIx Skin Cutaneous Melanoma SKCM 267 UNC-LCCC Illumina HiSeq 2000 Stomach adenocarcinoma STAD 41 BCCAGSC Illumina HiSeq 2000 Thyroid carcinoma THCA 373 UNC-LCCC Illumina HiSeq 2000 Uterine Corpus Endometrioid UCEC* 317 UNC-LCCC Illumina GA IIx Carcinoma *Single-end TCGA disease BAM files were downloaded from CGHub using its Gene Torrent Software

With the goal of supporting both single and paired-end data, 4,374 paired-end samples were processed with deFuse and 584 single-end samples with TopHat.

Broadly, the analysis pipeline consisted of 5 main steps: 1. Pre-process the raw data to obtain FASTQ files 2. Run fusion callers 3. Filter breakpoints to gene regions of interest 4. Annotate the breakpoints with the Oncomine transcript set and 5. Summarize and prioritize potentially interesting novel fusions.

The input to the fusion callers consisted of RNASeq reads in FASTQ format, which required conversion of the BAM file provided by TOGA to one or two FASTQ files for single or paired end data (respectively).

A custom SamToFastq converter was developed to generate FASTQ files from a TOGA BAM file. In addition to allowing conversion of all paired-end RNASeq TOGA BAMs systematically, the SamToFASTQ converter had other advantages over other conversion tools. First, it was written in C and compiled to run faster and reduce processing time. Second, it incorporated several validation steps to ensure proper mate pairing and consistent mate pair ordering in the output FASTQ files, both of which are input requirements for the fusion callers.

There were 3 cancer types (COAD, READ, UCEC) only available as single-end RNASeq data. For single-end BAM file conversion the program BamTools (github.com/pezmaster3l/bamtools) was used to generate FASTQ files.

Integration—FIG. 1 diagrams the relative levels of result filtering done by both callers. As part of the analysis “Level I” data was integrated—the output from TopHat-Fusion Post's potential_fusion.txt file and the output from deFuse's results.classify.tsv file. The integration steps involved converting the reported breakpoints to ones based on the genomic coordinate system and consolidation into a common file format.

Breakpoint Filtering—The ˜5.5 million predictions from the “Level I” output of the callers were filtered to only retain those calls where each breakpoint was either in the 5′UTR or CDS region of a RefSeq transcript (refGene circa Jul. 18, 2012, obtained from UCSC). This was done to enrich the predicted fusions for those containing functional gene regions. Breakpoints predicted to occur in intronic sequences were also excluded, resulting in a set of 423,587 predicted chimeras.

Breakpoint Annotation—For each pair of breakpoints, only one transcript per Entrez ID was retained. This ensured consistency in annotating breakpoints at the same location. However, predicted breakpoints at different locations for the same gene partners may still result in multiple transcripts representing a pair of genes—possible evidence of alternative transcripts.

Basic annotation from the callers was discarded, as it was based on the default annotation source of each respective caller. However, certain output fields from both TopHat and deFuse were retained to help prioritize the predicted fusions. Additionally, certain annotation properties that weren't explicitly reported by the callers were inferred from other caller properties.

Inferred Properties—Supporting and Spanning read counts were obtained from each caller and summarized in to Reads Span and Reads Span Support. The latter is a sum of reads spanning the fusion and those supporting the fusion. The breakpoint sequence reported by the callers was trimmed to include 50 bases on each side of the fusion and consolidated as Breakpoint Sequence. The fusion breakpoint is delineated by a “I”. Since neither of the callers provides a definitive ‘5-prime’ or ‘3-prime’ flag, the relative 5′-3′ orientation of the fusion partners was inferred by combining a caller parameter with the gene strand annotation. A Valid Orientation field was labeled as “Y” if there was an inferred 5′ and 3′ partner for a given gene fusion call.

RepeatMasker Annotation—Each predicted breakpoint location was also annotated with RepeatMasker features in the neighborhood of the breakpoint. This was done to identify breakpoints in highly repetitive genomic regions, where alignment errors were likely to affect the prediction of the chimeric transcript. For each fusion prediction, a RepeatMasker Overlap field was set to 1 if either of the breakpoint flank sequences overlaps with a RepeatMasker element by 12 or more bases. The frequency of overlapping fusion calls is used in the Oncomine Prioritization described below such that gene fusion predictions with a lower frequency of overlap are considered higher quality.

Fusion Exon Expression Imbalance—Recurrent Oncomine Priority Fusions were vizualized using RNASeq exon expression data downloaded using the GDAC Firehose tool to provide secondary evidence of true positive fusion events by searching for exon expression imbalance before and after the breakpoint call. Specifically, if the 3′ partner's expression is impacted by the 5′ partner's promoter region, then exon expression should increase post the predicted breakpoint. This effect is especially visible when viewing fused versus non-fused patient samples.

RPKM RNASeq values are listed for each patient as Gene Annotation Format (GAF) features corresponding to a composite of UCSC exons from several different gene definitions including Refseq. Compendia processed fusion breakpoints were mapped to the GAF features. 80.8% of the 396,298 Refseq exons map perfectly to GAF features in the plot shown below. The Refseq exon and GAF feature pair that resulted in the largest overlap was selected and reported on.

A value called rg_pct provides a metric of the mapping quality of a given Refseq exon with a GAF feature based on the following formula: rg_pct=overlap/length_(refseq)*overlap/length_(GAF feature)

Mappings with an rg_pct value of 1 overlap perfectly, while values less than 1 indicate the refseq exon or GAF feature did not map to the exact same genomic regions and the RPKM value may be suspect. RNASeq V2 data was selected for all diseases except OV, STAD, and LAML due to disease coverage shown in the barplot.

Fusion exon expression was manually reviewed for expression imbalance of a subset of Oncomine Priority fusions meeting the following criteria: 1. Recurrent Oncomine Priority Fusions 2. Oncomine Priority Fusions that are listed in the Mitelman Database 3. One fusion partner is an Oncomine Gain of Function Oncogene and involved in at least 3 Oncomine Priority Fusions and 4. One fusion partner is listed in the Sanger Cancer Gene Census (www.sanger.ac.uk/genetics/CGP/Census) and involved in at least 3 Oncomine Priority Fusions.

A total of 994 gene fusions meet these criteria and were manually reviewed for exon expression imbalance by assigning a “supported”, “refuted”, “neutral” or “not tested” rating to the gene fusion call.

Experts used the following criteria to assign ratings: Supported: Fused samples had a highly expressed 3′ fusion partner post-breakpoint such that fused samples were outliers of the patient population. Prior to the breakpoint, the 3′ partner's expression should be low compared to post-breakpoint. Refuted: Extremely low average expression of the 5′ partner (<5 RPKM) or average expression of one partner is much lower than the other (˜1/10). Neutral: Neither Support or Refute criteria are met. Fusions that were not manually reviewed were assigned a rating of Not Tested.

Fusion Summarization—Fusions were summarized within a disease based on the occurrence of unique gene pairs, and based on the occurrence of individual genes, possibly with multiple partners.

Fusion-Level Summary—For a unique fusion pair (unique by Entrez ID pair), the number of samples within a disease with at least one prediction of that fusion by either caller is the Fused Sample Count. Since multiple breakpoints for the same pair of genes may be reported in one sample and across the samples, the number of unique fusion pairs within each disease represented by the 424K+ fusion calls was 49,588. Table 10 shows the properties that were summarized for a given fusion partner pair across the individual predictions:

TABLE 10 Property Summary Method DEFUSE_EVERSION % of total fusion calls = ‘Y’ DEFUSE_VALID_ORIENTATION % of total fusion calls = ‘Y’ DEFUSE_NUM_MULTI_MAP % of total fusion calls > 0 TOPHAT_VALID_ORIENTATION % of total fusion calls = ‘Y’ 3P/5P_REPEATMASKER_OVERLAP % of total fusion calls = 1 The Adjacent flag is set for a fusion if the genes are <1 Mb apart on the genome and the defuse_eversion flag is set in ≤75% of the individual fusion prediction for these fusion partners.

Mitelman Cross-reference—Individual unique fusion pairs were cross-referenced to the Mitelman database of genomic aberrations (cgap.nci.nih.gov/Chromosomes/Mitelman downloaded Feb. 25, 2013). The match was done based on gene names and not disease type. Therefore, gene fusions reported in Mitelman in a certain disease may have occurred in a different disease type in the TCGA datasets. Gene fusions summarized at the gene level were cross-referenced to the Mitelman database based on gene name. Thus, there is more potential for the gene as reported in Mitelman to be of different histology or altogether different aberration type (for example a large chromosome-level deletion instead of a fusion) than the predicted unique fusion pairs.

Normal Sample Fusion Blacklist—To reduce the number of false positive fusions, 344 paired-end normal samples were processed across 10 diseases using the same deFuse pipeline described above. A total of 56,579 total fusion calls consisting of 6,024 unique fusions were observed. Of the 49,588 unique gene fusion events, 11,801 of these calls were observed in normal samples. These normal sample fusion calls were used to generate a blacklist and thereby remove these false positives from the Oncomine Priority gene fusions.

Paralogous Fusion Partner Blacklist—A blacklist of fusions between paralogous gene family members was assembled using two strategies: 1) manually inspecting high frequency fusion partner gene names and 2) comparing the first 3 characters of all Priority Fusion partner gene names. In the latter strategy, fusion partners were verified to be “paralogous” using HomoloGene, Ensembl, and SIMAP before inclusion in the final blacklist. This blacklist consists of 375 unique paralogous gene fusions and was used to remove false positives from the Oncomine Priority gene fusions.

Example 2: NGS Mutation Methods for Identifying Gene Variants Associated with Cancer

The goal of the data integration for gene variants was to create the most complete set of mutation data currently available from the TOGA.

Data Sources—For this release, the following were integrated: TOGA mutation data from the Broad GDAC Mutation_Packager 2013_02_22 stddata build, Level 2 (public, experimentally un-validated) data available from the TOGA DCC as of Mar. 1 2013, and, for prostate adenocarcinoma, mutation data generated by Compendia from TOGA primary data.

Compendia (CBI) Mutation Calls—There was concern that the prostate adenocarcinoma mutation calls available from TOGA were of low quality and resulted in false-positive ‘Gain of Function’ predictions. Therefore, all calls for this disease were sourced from Compendia's own mutation calling pipeline, which closely parallels the process used by the TOGA cancer type working groups to generate the publically-available mutation calls.

TABLE 12 Data Source Selection Mutation TCGA Packager DCC Cancer Type Disease (2013 Feb. 22) (2013 Mar. 1) Compendia TOTAL Bladder Urothelial BLCA 28 Carcinoma Breast Invasive BRCA 772 Carcinoma Cervical Squamous CESC 39 Cell Carcinoma and Endocervical Adenocarcinoma Colon COAD 153 Adenocarcinoma Glioblastoma GBM 290 Multiforme Head and Neck HNSC 306 Squamous Cell Carcinoma Kidney Renal Clear KIRC 293 Cell Carcinoma Kidney Renal Papillary KIRP 100 Cell Carcinoma Acute Myeloid LAML 196 Leukemia Brain Lower Grade LGG 169 Glioma Lung Adenocarcinoma LUAD 379 Lung Squamous Cell LUSC 178 Carcinoma Ovarian Serous OV 316 Cystadenocarcinoma Pancreatic PAAD 34 Adenocarcinoma Prostate PRAD 170 Adenocarcinoma Rectal READ 68 Adenocarcinoma Skin Cutaneous SKCM 252 Melanoma Stomach STAD 136 Adenocarcinoma Thyroid Carcinoma THCA 323 Uterine Corpus UCEC 235 Endometrioid Carcinoma 4,437

Data Cleaning—some simple clean-up operations were performed to remove duplicate mutation records present in the source data. Duplicate mutations from various tumor/normal aliquots pairs of the same patient sample were removed. A total of 25 “ultra-mutator” samples (mutation count of >5,000 per sample) were also excluded from the downstream analysis pipelines. In certain diseases, such as uterine corpus endometrioid carcinoma, several highly-mutated samples may dominate the overall mutation counts and dilute the results of mutation recurrence analysis necessary for the Compendia mutation and gene classification scheme.

Mutation Annotation: A. Compendia Annotation—Compendia's approach to defining mutations relied on accurate variant annotation hence; the mutations were re-annotated using a standard annotation pipeline which ensured that mutations across disease types were evaluated consistently and were subject to common interpretation during the nomination of potential oncogenes or tumor suppressor genes.

Mutations obtained from TCGA were processed by Compendia according to the following general steps: 1. Each mutation was first re-annotated using the Compendia transcript set. Successfully annotated mutations received Compendia-derived annotation, while the rest retain annotation obtained from the TCGA. Annotation includes: Variant classification, Variant position, Variant change. 2. Redundant annotations of a mutation in multiple transcripts were removed. 3. Mutations located outside of gene regions of interest were removed. 4. Mutations without a valid gene Entrez ID were removed.

“Mutation” is defined herein as a specific change at a genomic location, i.e.: Chromosome, start, stop, reference base, alternate base, variant type (SNP, INS, DEL) etc.

“Annotation” is defined herein as a transcript-specific set of properties that describe the effect of the mutation, i.e.: Gene, transcript, variant classification, variant change, variant codon position, etc.

In the Mutation Annotation step, the mutations obtained from TOGA were re-annotated against a standard transcript set compiled by Compendia. This transcript set included RefGene transcripts from hg18 and hg19 genome builds, obtained from UCSC.

Each mutation was individually mapped against a contig in the CBI Transcript Set within the specified genome build. SNP mutations were mapped directly to their start location, while for small insertion (INS) and deletion (DEL) mutations a position of interest is selected for mapping.

For a mutation successfully mapped to a transcript, the CBI mutation annotation was inferred with respect to that transcript. For mutations that fail to map, the more limited TOGA annotation was retained, and a variant position for Hotspot calculations was constructed based on the genomic coordinate.

Below is a description of the criteria used in annotating the mutations that map to the CBI Transcript Set:

Variant Classification:

For each mutation successfully mapped to a transcript, the variant classification was inferred using the location and the sequence variant type of the mutation. This approach identified the following main mutation variant classifications:

TABLE 13 main mutation variant classifications: Variant Classification Transcript Region Splice_Site exon or intron 3′UTR, 5′UTR UTR exon Intron intron Missense, Nonsense, Nonstop, Silent coding exon Frame_Shift_Ins/Del coding exon In_Frame_Ins/Del coding exon Non_Coding_Exon exon of a non-coding gene

Variant Position:

The variant position of a mutation is the location used to identify genes with Hotspot mutations, which are mutations of a certain classification that are observed at the same location in multiple tumor samples. To effectively identify recurrence and define a hotspot for each mutation, a mutation spot identifier was constructed that encompassed the mutation position, the identity of the amino acid or base affected, and the variant classification. Mutations that occurred at the same location irrespective of the specific base change they generated were aggregated. Therefore, only the reference base or amino acid was used to define the variant position. This ensured that mutations affecting the same codon or genomic position would be counted towards a possible hotspot, even if the alternate alleles they generated were different. For example, for a given gene, missense mutations V600E, V600F and V600G would all have a variant position of V600 and would thus be aggregated together when identifying hotspot mutations. When the amino-acid level position was not available, the RNA-level or genomic-level position was utilized.

For mutations that do not map to the CBI Transcript Set, and hence do not have a transcript-based location, the genomic location (start position) and the reference nucleotide (reference allele) was used as the variant position irrespective of the coding region or splice site proximity. The TOGA-annotated variant classification was then added as a suffix. The variant change (see below) for these mutations was not defined.

Variant Change:

The variant change provides HGVS-like information about the alternate allele change of the mutation (e.g. V600E). For SNP mutations in the coding region, the variant change was a full HGVS protein-level sequence variant description, indicating the alternate amino acid. For SNPs outside of the coding region, the alternate allele nucleotide base was provided. For mutations that do not map to the CBI Transcript Set, the variant classification from TOGA was retained.

Transcript Filtering:

To avoid retrieving multiple transcripts, and hence, multiple annotations for a single mutation within a gene, only one transcript per mutation per gene (unique Entrez ID) were kept. If a mutation mapped to several transcripts of a gene, only one was chosen. However, if a mutation mapped to several genes, then only one transcript per gene was selected. It was thus possible for a mutation to receive two different annotations, but only if they stemmed from transcripts with different Entrez IDs. In effect, any mutation of the same variant classification at the same genomic location was always assigned to the same transcript, and hence would be in the same frame of reference when computing recurrence for hotspot identification.

Gene Region Filtering:

All mutations were further filtered by variant type and class to avoid including mutations of minor interest to gene function analysis. Mutations were filtered out that were not resolved to a gene region, either because they fell significantly far outside of a transcript, or because they were in a location not associated with a Ref Seq gene. These mutations were evident either by their lack of gene identifier, or membership in the following variant classes: Intron, 5′Flank, IGR, and miRNA. Mutations were also filtered out with variant type of DNP, TNP, ONP, Complex_substitution, and Indel, as their annotation was not supported by the pipeline

Classifying Mutations as Hotspot, Deleterious, or Other—The next step in the analysis pipeline identified recurring mutations in multiple samples based on their variant position, and categorized them into Hotspot, Deleterious or Other variant categories. For this step, and the subsequent frequency calculations, mutations for each disease type were processed independently. Only mutations of the same variant classification were tallied together, so, for example, a missense mutation and a silent mutation at the same position was counted separately.

To identify driver events, each mutation for a given Entrez Gene Id was categorized as “Deleterious” or “Hotspot”. A mutation was deemed ‘recurrent’ if it was observed in the same variant position in 3 or more tumor samples. A mutation belonged to the “Hotspot” variant category if it was recurrent and was annotated with one of the following variant classifications: In-frame insertion/deletion, Nonstop, Missense, Non_Coding_Exon. A mutation belonged to the “Deleterious” category if it was: annotated with one of the following variant classifications: Frame shift insertion/deletion, Nonsense. A mutation was considered in the “Other” variant category if it did not fit the above criteria.

Nominating “Gain of Function” and “Loss of Function” Genes—Individual genes were classified into predicted functional classes, namely “Gain of Function”, “Recurrent Other”, and “Loss of Function” to reflect their relative enrichment in potential activating or deleterious mutations.

Frequency of Mutations:

Mutation frequencies for each gene were calculated with respect to a given variant classification and variant category across all samples within a disease type. Overall mutation frequency for a gene within a disease was calculated by combining all the mutations.

Mutation Significance:

The Hotspot p-values for each gene within a disease were calculated by selecting the most recurrent mutation m and using sampling to determine the probability p of observing r or more mutations at that position. More specifically: p=100,000−Σ_(m=1) ^(r-1)c_(m)/100,000, where c_(m) is the count of replicates with maximum multiplicity m. P-values for transcripts with a maximum multiplicity of one are defined as 1.0. P-value for transcripts with a maximum multiplicity that is never observed is defined as 1 e-5.

Hotspot Q-values were calculated within each disease by counting the number of transcripts mutated at least once (N) and calculating the rank of each p-value. The q-value for a given p-value is then Q=p*N/rank.

To assess whether a gene was significantly enriched for deleterious mutations compared with other genes, given the background mutation rate, Fisher's exact test was performed comparing the deleterious mutation frequency of the gene in question to that of other genes. Nonsense mutations, frame shift insertions and frame shift deletions were classified as deleterious mutations, while mutations of any other type (missense, etc., but non-intergenic) counted as others.

Deleterious Q-values were calculated within each disease, by counting the number of genes with deleterious mutations (N), and calculating the rank of each association. The q-value for a given p-value was then Q=p*N/rank.

Gene Classification:

Once the mutations were classified, individual genes were nominated to one of three classes—“Gain of Function,” “Loss of Function,” and “Recurrent Other.” The classification is based on the combination of relative frequencies and the significance of the mutations observed in the gene. The significance of the mutations per gene is assessed by a p-value. The classification scheme in FIG. 2 specifies the criteria for Gain of Function and Loss of Function genes.

A “Gain of Function” gene will have a relatively high frequency of Hotspot Missense mutations and a low frequency of Deleterious mutations, while a “Loss of Function” gene contains a large fraction of Deleterious mutations. “Recurrent Other” tend to contain recurrent insertion/deletion mutations, some of which—for example recurrent frame shift indels of 1 base—exhibit signs of potential false-positive calls that may arise from local alignment errors.

Pan-Cancer Analysis—To summarize mutations across diseases identical calculations were performed as for within-disease analyses, but without stratifying the mutation records by disease. For the pan-disease gene classification, the genes (unique by Entrez ID) were summarized across all cancer types.

Example 3: Diagnostic Assay for the Identification of Gene Fusions and/or Gene Variants in Cancer

Library Preparation

PCR Amplify Genomic DNA Targets

The disclosed variant and fusion polynucleotides can be detected by the sequencing of nucleic acids. This can be accomplished by next generation sequencing, the description of which follows. The source of the nucleic acid for next generation sequencing can include a Fresh-Frozen Paraffin-Embedded (FFPE) sample.

A multiplex polymerase chain reaction is performed to amplify 384 individual amplicons across a genomic DNA sample. A pool of greater than 32,000 primers is developed covering more than 100 gene variants or fusion polynucleotides. Each primer in the primer pool was designed to contain at least one uridine nucleotide near the terminus of each primer. Each primer is also designed to selectively hybridize to, and promote amplification, by forming a primer pair, with a specific gene, gene variant, or fusion polypeptide of a nucleic acid sample.

To a single well of a 96-well PCR plate is added 5 microliters of the Primer Pool containing 384 primer pairs at a concentration of 15 μM in TE, 10-50 ng genomic DNA and 10 microliters of an amplification reaction mixture (2× AmpliSeq HiFi Master Mix) that can include glycerol, dNTPs, and Platinum® Taq High Fidelity DNA Polymerase (Invitrogen, Catalog No. 11304) to a final volume of 20 microliters with DNase/RNase Free Water (Life Technologies, CA, Part No. 600004).

The PCR plate is sealed and loaded into a thermal cycler (GeneAmp® PCR system 9700 Dual 96-well thermal cycler (Life Technologies, CA, Part No. N8050200 and 4314445)) and run using the following temperate profile to generate the preamplified amplicon library.

An initial holding stage is performed at 98° C. for 2 minutes, followed by 16 cycles of denaturing at 98° C. for 15 seconds and an annealing and extending stage at 60° C. for 4 minutes. After cycling, the preamplified amplicon library is held at 4° C. until proceeding to the purification step outlined below.

Purify the Amplicons from Input DNA and Primers

Two rounds of Agencourt® AMPure® XP Reagent (Beckman Coulter, CA) binding, wash, and elution at 0.6× and 1.2× volume ratios are found to remove genomic DNA and unbound or excess primers. The amplification and purification step outlined herein produces amplicons of about 100 bp to about 600 bp in length.

In a 1.5 ml LoBind tube (Eppendorf, Part No. 022431021), the preamplified amplicon library (20 microliters) is combined with 12 microliters (0.6× volumes) of Agencourt® AMPure® XP reagent (Beckman Coulter, CA). The bead suspension is pipetted up and down to thoroughly mix the bead suspension with the preamplified amplicon library. The sample is then pulse-spin and incubated for 5 minutes at room temperature.

The tube containing the sample is placed on a magnetic rack such as a DynaMag™-2 spin magnet (Life Technologies, CA, Part No. 123-21 D) for 2 minutes to capture the beads. Once the solution cleared, the supernatant is transferred to a new tube, where 24 microliters (1.2× volume) of AgenCourt® AMPure® XP beads (Beckman Coulter, CA) is added to the supernatant. The mixture is pipetted to ensure that the bead suspension is mixed with the preamplified amplicon library. The sample is then pulse-spun and incubated at room temperature for 5 minutes. The tube containing the sample is placed on a magnetic rack for 2 minutes to capture the beads. Once the solution clears, the supernatant is carefully discarded without disturbing the bead pellet. The desired preamplified amplicon library is then bound to the beads. Without removing the tube from the magnetic rack, 200 microliters of freshly prepared 70% ethanol is introduced into the sample. The sample is incubated for 30 seconds while gently rotating the tube on the magnetic rack. After the solution clears, the supernatant is discarded without disturbing the pellet. A second ethanol wash is performed and the supernatant discarded. Any remaining ethanol is removed by pulse-spinning the tube and carefully removing residual ethanol while not disturbing the pellet. The pellet is air-dried for about 5 minutes at room temperature.

Once the tube is dry, the tube is removed from the magnetic rack and 20 microliters of DNase/RNase Free Water is added (Life Technologies, CA, Part No. 600004). The tube is vortexed and pipetted to ensure the sample is mixed thoroughly. The sample is pulse-spun and placed on the magnetic rack for two minutes. After the solution clears, the supernatant containing the eluted DNA is transferred to a new tube.

Phosphorylate the Amplicons

To the eluted DNA (˜20 microliters), 3 microliters of DNA ligase buffer (Invitrogen, Catalog No. 15224041), 2 microliters dNTP mix, and 2 microliters of FuP reagent are added. The reaction mixture is mixed thoroughly to ensure uniformity and incubated at 37° C. for 10 minutes.

Ligate Adapters to the Amplicons and Purify the Ligated Amplicons

After incubation, the reaction mixture proceeds directly to a ligation step. Here, the reaction mixture now containing the phosphorylated amplicon library is combined with 1 microliter of A/P1 Adapters (20 μm each)(sold as a component of the Ion Fragment Library Kit, Life Technologies, Part No. 4466464) and 1 microliter of DNA ligase (sold as a component of the Ion Fragment Library Kit, Life Technologies, Part No. 4466464), and incubated at room temperature for 30 minutes.

After the incubation step, 52 microliters (1.8× sample volume) of AgenCourt® AMPure® Reagent (Beckman Coulter, CA) is added to the ligated DNA. The mixture is pipetted thoroughly to mix the bead suspension with the ligated DNA. The mixture is pulse-spun and incubated at room temperature for 5 minutes. The samples undergo another pulse-spin and are placed on a magnetic rack such as a DynaMag™-2 spin magnet (Life Technologies, CA, Part No. 123-21D) for two minutes. After the solution clears, the supernatant is discarded. Without removing the tube from the magnetic rack, 200 microliters of freshly prepared 70% ethanol is introduced into the sample. The sample is incubated for 30 seconds while gently rotating the tube on the magnetic rack. After the solution clears, the supernatant is discarded without disturbing the pellet. A second ethanol wash is performed and the supernatant is discarded. Any remaining ethanol is removed by pulse-spinning the tube and carefully removing residual ethanol while not disturbing the pellet. The pellet is air-dried for about 5 minutes at room temperature.

The pellet is resuspended in 20 microliters of DNase/RNase Free Water (Life Technologies, CA, Part No. 600004) and vortexed to ensure the sample is mixed thoroughly. The sample is pulse-spun and placed on the magnetic rack for two minutes. After the solution clears, the supernatant containing the ligated DNA is transferred to a new Lobind tube (Eppendorf, Part No. 022431021).

Nick Translate and Amplify the Amplicon Library and Purify the Library

The ligated DNA (˜20 microliters) is combined with 76 microliters of Platinum® PCR SuperMix High Fidelity (Life Technologies, CA, Part No. 12532-016, sold as a component of the Ion Fragment Library Kit, Life Technologies, Part No. 4466464) and 4 microliters of Library Amplification Primer Mix (5 μM each)(Life Technologies, CA, Part No. 602-1068-01, sold as a component of the Ion Fragment Library Kit, Life Technologies, Part No. 4466464), the mixture is pipetted thoroughly to ensure a uniformed solution. The solution is applied to a single well of a 96-well PCR plate and sealed. The plate is loaded into a thermal cycler (GeneAmp® PCR system 9700 Dual 96-well thermal cycler (Life Technologies, CA, Part No. N8050200 and 4314445)) and run on the following temperate profile to generate the final amplicon library.

A nick-translation is performed at 72° C. for 1 minute, followed by an enzyme activation stage at 98° C. for 2 minutes, followed by 5-10 cycles of denaturing at 98° C. for 15 seconds and an annealing and extending stage at 60° C. for 1 minute. After cycling, the final amplicon library is held at 4° C. until proceeding to the final purification step outlined below.

In a 1.5 ml LoBind tube (Eppendorf, Part No. 022431021), the final amplicon library (˜100 microliters) is combined with 180 microliters (1.8× sample volume) of Agencourt® AMPure® XP reagent (Beckman Coulter, CA). The bead suspension is pipetted up and down to thoroughly mix the bead suspension with the final amplicon library. The sample is then pulse-spun and incubated for 5 minutes at room temperature.

The tube containing the final amplicon library is placed on a magnetic rack such as a DynaMag™-2 spin magnet (Life Technologies, CA, Part No. 123-21D) for 2 minutes to capture the beads. Once the solution clears, the supernatant is carefully discarded without disturbing the bead pellet. Without removing the tube from the magnetic rack, 400 microliters of freshly prepared 70% ethanol is introduced into the sample. The sample is incubated for 30 seconds while gently rotating the tube on the magnetic rack. After the solution clears, the supernatant is discarded without disturbing the pellet. A second ethanol wash is performed and the supernatant is discarded. Any remaining ethanol is removed by pulse-spinning the tube and carefully removing residual ethanol while not disturbing the pellet. The pellet is air-dried for about 5 minutes at room temperature.

Once the tube is dry, the tube is removed from the magnetic rack and 20 microliters of Low TE was added (Life Technologies, CA, Part No. 602-1066-01). The tube is pipetted and vortexed to ensure the sample is mixed thoroughly. The sample is pulse-spin and placed on the magnetic rack for two minutes. After the solution clears, the supernatant containing the final amplicon library is transferred to a new Lobind tube (Eppendorf, Part No. 022431021).

Assess the Library Size Distribution and Determine the Template Dilution Factor

The final amplicon library is quantitated to determine the library dilution (Template Dilution Factor) that results in a concentration within the optimized target range for Template Preparation (e.g., PCR-mediated addition of library molecules onto Ion Sphere™ Particles). The final amplicon library is typically quantitated for downstream Template Preparation procedure using an Ion Library Quantitation Kit (qPCR) (Life Technologies, Part No. 4468802) and/or a Bioanalyzer™ (Agilent Technologies, Agilent 2100 Bioanalyzer) to determine the molar concentration of the amplicon library, from which the Template Dilution Factor is calculated. For example, instructions to determine the Template Dilution Factor by quantitative real-time PCR (qPCR) can be found in the Ion Library Quantitation Kit User Guide (Life Technologies, Part No. 4468986), hereby incorporated by reference in its entirety.

In this example, 1 microliter of the final amplicon library preparation is analyzed on the 2100 Bioanalyzer™ with an Agilent High Sensitivity DNA Kit (Agilent Technologies, Part No. 5067-4626) to generate peaks in the 135-205 bp size range and at a concentration of about 5×10⁹ copies per microliter.

Proceed to Template Preparation

An aliquot of the final library is used to prepare DNA templates that are clonally amplified on Ion Sphere™ Particles using emulsion PCR (emPCR). The preparation of template in the instant example is prepared according to the manufacturer's instructions using an Ion Xpress Template Kit (Life Technologies, Part No. 4466457), hereby incorporated by reference in its entirety. Once template-positive Ion Sphere Particles are enriched, an aliquot of the Ion Spheres are loaded onto an Ion 314™ Chip (Life Technologies, Part No. 4462923) as described in the Ion Sequencing User Guide (Part No. 4467391), hereby incorporated in its entirety, and subjected to analysis and sequencing as described in the Ion Torrent PGM Sequencer User Guide (Life Technologies, Part No. 4462917), hereby incorporated in its entirety.

Example 4: Oncomine NGS Integrative Analysis Methods to Identify Genetic Events Associated with Clinical Outcomes

The Oncomine NGS Integrative Analysis was designed to bring together the largest possible set of core NGS data to enable scientific workflows that interrogate relationships across data types and diseases, summarizing the analyses at multiple biological levels of abstraction, such as genes and pathways.

Data Sources (Oncomine is available from Life Technologies/Compendia Biosciences—Ann Arbor, Mich. and www.oncomine.org).

The data for the Integrative Analysis was taken from the below sources:

Fusions: Oncomine driver fusions

Mutations: Oncomine pan-cancer driver mutations

CNVs: Peak amplification and deletion data derived from Oncomine-processed copy number data

DNA: Oncomine-processed DNA-seq continuous data

RNA: Normalized gene-level RNAseq continuous data

Clinical: Oncomine-curated clinical and outcome metadata

Pathways: Oncomine pathway definitions

Fusions Data and Filtering

Fusion data for integrative analysis was obtained from Oncomine NGS Fusion data. Oncomine Prioritized Fusion is a priority scheme developed at Compendia to capture attributes of known true positive fusion events and characterize a subset of observed gene fusions as high-confidence priority fusions. Criteria used to define priority fusions include: valid 5′ to 3′ orientation, non-adjacent fusion partners, uniquely mapping spanning reads, non-paralogous fusion partners, not observed in normal tissue, and non-overlapping with redundant regions in the genome.

Fusions were included and considered driver fusions if they were called by deFuse or Tophat, had exon expression evidence that was “supported” or “neutral” and met one of the following 4 criteria:

Oncomine Prioritized Fusion+Recurrent

Oncomine Prioritized Fusion+Mitelman Annotated

Oncomine Prioritized Fusion+One partner is an Oncomine Gain of Function gene involved in 3 or more Pan-Disease Priority Fusions

Oncomine Prioritized Fusion+One partner is a Sanger Oncogene (goo.gl/JQBw9) involved in 3 or more Pan-Disease Priority Fusions

Mutations Data and Filtering

Mutation data for Integrative Analysis was obtained from Oncomine NGS Mutation data. Individual genes are classified into predicted functional classes, namely “Gain of Function” and “Loss of Function” to reflect their relative enrichment in potential activating or deleterious mutations. This classification is based on the combination of relative frequencies and the significance of the mutations observed in the gene assessed by a p-value. A “Gain of Function” gene will have a relatively high frequency of Hotspot Missense mutations and a low frequency of Deleterious mutations, while a “Loss of Function” gene contains a large fraction of Deleterious mutations.

Copy Number Segmentation and Quantification

DNA copy number data for each TCGA sample was obtained from Oncomine.

Measurements from multiple reporters for a single gene were averaged.

Minimum Common Region (MCR) Peak Generation

In genes that were recurrently amplified (4 or more copies) or deleted (1 or less copy), peaks were identified independently in 25 cancer types by applying MCR analysis on Oncomine clinical samples. To define peaks, contiguous genomic regions with multiple genes that were significantly aberrant (common regions) were identified first. In every common region, a Peak is defined as one or more genes whose aberrant sample count meets or exceeds a peak threshold. In every cancer, common regions are defined as regions whose aberrant sample count meet or exceed a common region threshold. The baseline, average number of aberrant samples observed across all genes, is calculated for every arm of every chromosome in every cancer.

mRNA Gene Expression

Expression data was obtained from the Broad GDAC's TCGA Standard Data.

Clinical Data Curation

Patient clinical data was obtained from TCGA and curated by Compendia. Curated data types included demographics, major clinical and histological disease subtypes, and clinical outcome data. All properties were standardized to be consistent across the diseases.

Construction of Clinically Relevant Subsets

Curated clinical data obtained from TCGA and Oncomine NGS data was used and the rules in Table 14 were applied to define the Clinical Subsets:

TABLE 14 Rules to define the Clinical Subsets Disease Clinical Subtype Source Rules Invasive Breast Triple Negative Phenomic Data ERBB2 Status = ERBB2 Negative Carcinoma Estrogen Receptor Status = Estrogen Receptor Negative Progesterone Receptor Status = Progesterone Receptor Negative ER Positive Phenomic Data Estrogen Receptor Status = Estrogen Receptor Positive ER Positive and Phenomic Data Estrogen Receptor Status = Estrogen Receptor HER2 Negative Positive ERBB2 Status = ERBB2 Negative Gastric Hyper-Mutator Oncomine NGS Data Patient Mutation Count >= 400 Adenocarcinoma Lung KRAS Mutation (No Oncomine NGS Data Oncomine Mutation Classification = Hotspot Adenocarcinoma ALK Fusion and No EGFR Mutation) Triple Negative Oncomine NGS Data No EGER Mutation (AND) No KRAS Mutation (AND) No ALK Fusion Rectal KRAS Mutation Oncomine NGS Data Oncomine Mutation Classification = Hotspot Adenocarcinoma KRAS Mutation, Oncomine NGS Oncomine Mutation Classification = Hotspot Stage 3 or 4 Data/ (AND) Phenomic Data Stage = Stage III (OR) Stage IV KRAS Wildtype Oncomine NGS Data No KRAS Mutation

Pathways

Manually curated Compendia pathway definitions were used to summarize gene-level aberrations in the integrative analysis. The pathways represent clinically relevant pathway modules, and several modules may cover a major biological pathway, and a single gene may be present in one or more pathway module definitions.

Data Integration

The diagram in FIG. 3 summarizes the data flow that integrates the various data types into a Genetic Event Database (GEDB). All further analyses are conducted using the GEDB. The process has 4 main steps.

Map the data to the internal IA gene and patient dimension

Define events and driver events in each data type

Roll-up individual events to the gene and pathway level

Combine the events into the Genetic Events Database.

Gene and Patient Dimensions

A single gene and patient dimension was constructed which encompassed all patients and genes measured across all disease and data types. The genes and patients were given internal identifiers, and all data in the IA was referenced against these identifiers for gene name and patient barcode consistency. The unique identifier for a gene is the gene Entrez ID. The unique identifier for a patient is the TCGA Patient Barcode (first 12 digits of the TCGA barcode).

Driver Event Definition

Mutation, fusion and copy number events are defined based on the following criteria for genomic events:

Fusions: Oncomine recurrent priority fusions

Mutations: Oncomine driver mutations from pan cancer driver genes

CNVs: CBI identified peaks, and gene amp/del within peaks

Genetic Event Definition and Roll-up

A genetic event is a genomic aberration, representing either an individual mutation, fusion, or copy number event, or a combination of events at the gene or pathway level. The events are ‘rolled-up’ according to the flowchart shown in FIG. 4 . When multiple events are combined to construct rolled up events, the set of measured patients for the rolled up event becomes the intersection of the patients measured for all 3 data types. Patients positive are only included if fully measured.

Analyses

Once all the driver genetic events are constructed, a set of analyses is performed on each genetic event, calculating frequencies, associations and relationships within diseases (and pan-cancer where appropriate). The following are short descriptions of each analysis:

Frequency

Frequency is the occurrence of a driver event among the patients in which it was measured. Frequencies are calculated within disease and pan-cancer.

Clinical Association Analysis

Each driver event is tested for association against a set of available clinical subtypes. Each association is tested using a Fischer's exact test by comparing the occurrences of the genetic event in patients of one clinical subtype versus another. For example a Loss of Function mutation may be tested for over-representation in Smokers versus Non-Smokers, or in Stage I versus Stage II lung cancer. A total of 136 subtype pairs are tested against each event, the properties that define the subtypes are listed below (some properties may be disease-specific). At least 4 patients total, with at least 1 patient in each class are required to perform the test.

Clinical Outcome Analysis

Each driver event is tested for association with clinical outcome using log-rank test. Only the set of patients with available clinical data are used for the calculation, so the number of patients included in the test may be less than the number of patients measured for the driver event. At least 4 patients positive for driver event are required to perform the test. Survival time is presented in years, and individual alive/dead events are clearly marked on a Kaplan-Meier curve. P-values were corrected for multiple testing (q-values). Events with a q-value less than 0.1 were considered.

The results of the analysis are shown in Tables 15 and 39. In Tables 15 and 39, the columns provide the following information:

The “Subset” column provides the clinically relevant cancer type.

The p-value column is the p-value.

The q-value column is the corrected p-value. Events with q<0.1 are included in the table.

The no. positive column is the number of patients positive for an event type.

The Total no. of patients column is the total number patients assessed.

The Cytoband column is the chromsomal location of the gene(s).

The Genes (Entrez ID) column is a List of gene(s) and corresponding Entrez id.

The Druggable genes column indicates if any gene(s) are targets for drugs in active trials, approved, or otherwise commercially available.

The KM Evidence column provides the Kaplan-Meier evidence. The KM evidence indicates if the event type supports good or poor prognosis in the particular cancer type.

Tables 15 and 39 contains more than 100 gain-of-function mutations, loss-of-function mutations, in-peak gene amplification/deletions, and fusion events for various cancer types with a q<0.1. Gene(s) within each event and cancer type are included along with their chromosomal locations, druggability information and clinical outcome associations, as indicated in the column information above.

Example 5—Integrated Data Analysis

Oncomine NGS Integrated Analysis. The Oncomine NGS Integrative Analysis was designed to bring together the largest possible set of core integrated genomic and phenomic data to enable scientific workflows that interrogate relationships across data types and cancer types, summarizing the analyses at multiple biological levels of abstraction, such as genes and pathways.

Terminology:

Aberration—A genomic structural variation or alteration of DNA; Examples include: mRNA over/under-expression, copy number amplification/deletion, mutation, and gene fusion.

Driver—Aberration identified as a potential cancer driver by Oncomine methodology described in this document; examples include gain of function mutations, gene amplifications in a peak amplification region, or gene fusions

Roll-up—A summary of all mutation, fusion, or copy-number aberrations for the gene or pathway; Only patients measured for all three aberration types are included in the rolled-up.

Hotspot Mutation—A mutation that is recurrent (n≥3), and classified as either an in-frame insertion/deletion, nonstop or missense.

Patient null set—The set of patients measured for a genetic aberration.

Patient positive set—The set of patients harboring the genetic aberration.

Gene null set—The set of genes measured by the experimental platform used to assess the genetic aberration.

Mitelman—Database of Chromosome Aberrations and Gene Fusions in Cancer manually curated from literature (goo.gl/PnXMT).

RPKM—“Reads Per Kilobase per Million”; a method for RNASeq data quantification that normalizes for total read length and number of sequencing reads (Mortazavi et al. 2008).

RSEM—“RNA-Seq by Expectation Maximization” a method for RNASeq data quantification that estimates the best probable distribution of reads among the expected transcripts provides relative transcript abundances as a fraction of the total read pool. (Li and Dewey 2011)

Data Sources.

An effort was made to collect the largest overlapping set of data available for each sample. The data in this release of the NGS Integrative Analysis Browser was obtained from The Cancer Genome Atlas (TCGA), the Cancer Cell Line Encyclopedia (CCLE), COSMIC Cell Lines Project, and a number of research publications, either directly or after being subjected to Oncomine processing and analysis methods. Due to the uneven coverage of all data types across the source datasets, some cancer types have a greater number of patients covered in multiple data types.

The Oncomine NGS Mutations release used in the Integrative Analysis contained a number of hand-curated datasets obtained from NGS mutation studies in peer-reviewed publications. For a full list of publications that contributed mutation data to integrative analysis, please see the Oncomine NGS Mutations methods documentation. The following datasets contained multi-dimensional NGS data, providing both, mutations and copy number data. Copy number data for these datasets was processed in the same way as the copy number data obtained from TCGA.

Cell line data includes mutation, fusion, and copy number datasets. Cell line data was processed in the same way as the clinical tumor data—with mutation and fusion cell line data obtained from the Oncomine™ NGS Mutation and Oncomine™ NGS Fusion Power Tools, respectively. Copy number data for cell lines was processed using the standard Oncomine copy number pipeline. Although there were two disparate cell line datasets used—CCLE and COSMIC—our standardization of cell line disease types and names has enabled us to cross reference the two datasets and combine the CCLE copy number data, COSMIC mutation data and Oncomine fusions calls (based on CCLE RNASeq data). Therefore, numerous cell lines in this release have had their exomes systematically characterized for all three types of aberrations. Cell line data was summarized using the Oncomine cancer type definitions to be directly comparable to tumor data, although the summarization was performed separately for tumor and cell lines.

Phenomic Data

Clinical Patient Metadata Curation.

Patient clinical data was obtained from primary sources and curated by Compendia. Curated data types include demographics, major clinical and histological disease subtypes, and clinical outcome data. All cancer type-independent properties (such as age or survival) were standardized for consistency across cancer types. Certain disease stages were merged to obtain higher patient counts within a stage. For example, Stage Ia and Ib may be combined as Revised Stage I.

Following is the list of most populated properties and corresponding values captured by the curation process. Not all properties were available for all patients.

Property Name Property Value Age 10-14 Years 15-19 Years 20-29 Years 30-39 Years 30-39 Years 40-49 Years 50-59 Years 60-69 Years 70-79 Years 80-89 Years   90+ Years ERBB2 Status ERBB2 Negative ERBB2 Positive Estrogen Receptor Status Estrogen Receptor Negative Estrogen Receptor Positive FAB Subtype FAB Subtype M0 FAB Subtype M1 FAB Subtype M2 FAB Subtype M3 FAB Subtype M4 FAB Subtype M5 FAB Subtype M6 FAB Subtype M7 Gleason Score Gleason Score 10 Gleason Score 6 Gleason Score 7 Gleason Score 8 Gleason Score 9 Grade Grade 1 Grade 2 Grade 3 Grade 3-4 Grade 4 Hepatitis Virus Infection Status Hepatitis B Virus Positive Hepatitis C Virus Positive Human Papillomavirus Infection HPV Negative Status HPV Positive HPV Type 16 and 52 Positive HPV Type 16 Positive HPV Type 45 Positive HPV Type 58 Positive Metastatic Event Status Metastatic Event Microsatellite Status Microsateilite Instable Microsateilite Stable Overall Survival Status Alive Dead Overall Survival Status Alive (Detailed) Alive With Disease Alive Without Disease Dead Dead With Disease Dead Without Disease Patient Treatment Response Unknown Therapy Complete Response Unknown Therapy Partial Response Unknown Therapy Progressive Disease Unknown Therapy Stable Disease Progesterone Receptor Status Progesterone Receptor Negative Progesterone Receptor Positive Race/Ethnicity American Indian or Alaska Native Asian Black or African American Hispanic or Latino Native Hawaiian or Other Pacific White Recurrence Status Biochemical Recurrence No Biochemical Recurrence Recurrence Recurrence Status (Detailed) Local Recurrence Metastatic Recurrence Recurrence Revised M Stage M0 M1 Revised N Stage N0 N1 N2 N3 Revised Smoking Status Never Smoker Smoker Revised Stage FIGO Stage I FIGO Stage II FIGO Stage III FIGO Stage IV Stage I Stage II Stage III Stage IV Revised T Stage T T0 T1 T11 T12 T2 T21 T22 T3 T4 Sex Female Male *TCGA PAM50 Subtype Basal-like HER2-enriched Luminal A Luminal B Normal-like *TCGA RPPA Subtype Basal Her2 Luminal A Luminal A/B Reactive I Group Reactive II Group *TCGA Subtype Basal CIN Classical Invasive Mesenchymal MSI/CIMP Neural Primitive Proneural Secretory Metastatic Event Follow-up (Days) Time Overall Survival Follow-up (Days) Time Recurrence Follow-up Time (Days)

Properties prefixed by “TOGA” were obtained and curated from the TOGA publications that defined the molecular subtypes for invasive breast carcinoma, glioblastomasquamous cell lung carcinoma and colorectal cancers.

Genomic Event Data: Fusions Data Filtering.

Fusion data for the Integrative Analysis Browser was obtained from Oncomine NGS Fusion data released in November, 2013. Only fusions identified as Oncomine Priority Fusions were included in the Integrative Analysis Browser.

Oncomine Prioritized Fusion is a priority scheme developed at Compendia to capture attributes of known true positive fusion events and characterize a subset of observed gene fusions as high-confidence priority fusions. Criteria used to define priority fusions include: valid 5′ to 3′ orientation, non-adjacent fusion partners, uniquely mapping spanning reads, non-paralogous fusion partners, not observed in normal tissue, and non-overlapping with redundant regions in the genome.

The patient null set for the fusion data is the full set of patient tumor samples processed in the fusion analysis; data for only one tumor sample (preferably the primary, non-recurrent tumor) per patient was retained. The gene null set is the set of genes in RefGene as of May 2012. Fusions were included in the Integrative Analysis Browser if they were an Oncomine Priority Fusion, had exon expression evidence that was “supported” or “neutral”, and met one of the following criteria:

Recurrent (occurred in 2 or more patients)

Annotated in the Mitelman database of known structural variations

Contained a gene partner that is an Oncomine Gain of Function gene that is involved in 3 or more Pan-Disease Priority Fusions

Contained a gene partner that is a Sanger Oncogene (goo.gl/JQBw9) that is involved in 3 or more Pan-Disease Priority Fusions.

Mutation Data Filtering.

Mutation data for Integrative Analysis was obtained from Oncomine NGS Mutation data released in November, 2013. Only non-silent mutations in coding gene regions were included in the Integrative Analysis Browser.

The patient null set is the full set of patients processed in the mutation analysis; data for only one tumor sample (preferably the primary, non-recurrent tumor) per patient was retained. The gene null set is the set of genes in Ref Gene as of March 2012.

Mutations with the following variant classifications were not included in the Integrative Analysis Browser: Silent, 5′ UTR, 3′ UTR, RNA, Non-Coding Exon.

Calling Amplifications/Deletions.

DNA copy number data for each sample was obtained from the 2013 Q4 Oncomine Standard Data Build, in which all copy number data available from TCGA and the hand-curated publications as of October 2013 was standardized.

The patient null set for this analysis was the set of patients measured for copy number data as of October 2013 and the set of patients measured in the hand-curated publications. Data for only one tumor sample (preferably the primary, non-recurrent tumor) per patient was retained. The gene null set for this data was the Oncomine DNA Copy Number platform, based on RefSeq coordinates (hg18) provided by UCSC RefGene build July 2009, and measures 18,796 genes. Measurements from multiple reporters for a single gene were averaged.

The log₂ of the estimated copy value was used to make amplification/deletion (amp/del) calls, with cutoffs of >1.0 and <−1.0, respectively. No amp/del calls were made log₂ (estimated copies) that were ≥−1.0 or ≤+1.0.

Genomic Continuous Data: Copy Number Segmentation and Quantification.

DNA copy number data for each sample was obtained from the 2013 Q4 Oncomine Standard Data Build, in which all copy number data available from TCGA as of September 2013 and all copy number data from the hand-curated publications was standardized.

The patient null set for this analysis was the set of TCGA patients measured for copy number data as of October 2013 and the set of patients measured in the hand-curated publications. Data for only one tumor sample (preferably the primary, non-recurrent tumor) per patient was retained. The gene null set for this data was the Oncomine DNA Copy Number platform, based on RefSeq coordinates (hg18) provided by UCSC Ref Gene build July 2009, and measures 18,796 genes. Measurements from multiple reporters for a single gene were averaged.

Copy number data was segmented and quantified using the standard Oncomine processing pipeline. Segmentation is a method used to identify contiguous regions of amplification or deletion. These regions or “segments” can include multiple genes or single genes. A copy number value is computed for each segment based on the mean value for the reporters contained in the segment. Genes are mapped to segments and assigned a value. This gene level data is then reported. Please see the Oncomine DNA Processing Pipeline White Paper for more information.

mRNA Expression Data.

Expression data was obtained from the Broad GDAC's TOGA Standard Data build from September, 2013.

The patient null set for this data was the set of patients with available RNASeq data in the Broad GDAC 2013_08_09 stddata build; data for only one tumor sample (preferably the primary, non-recurrent tumor) per patient was retained. The gene null set for this data was different per disease and corresponded to the TOGA Gene Annotation Files (GAFs) used for the RNASeq quantification.

The TOGA currently employs two methods of RNASeq quantification—V1 (RPKM) and V2 (RSEM)—which are not directly numerically comparable. To avoid a potentially inaccurate numerical conversion, we use data from a single quantification method on a per-disease basis, choosing the format based on maximal coverage. In line with efforts by the TOGA to process (and re-process) all available RNASeq data using RSEM (V2), RSEM (V2) data was available for most samples. An exception is Gastric Adenocarcinoma where RPKM (V1) data was used. Normalized, gene-level quantification values were obtained for both RSEM and RPKM and converted to log₂ values (minimum non-zero RPKM or RSEM values were set at −12). A gene was considered to be expressed if it had a log₂ value>−12.

Oncomine Driver Reference Data: Minimum Common Region (MCR) Peak Generation and Gene Selection.

In order to identified cancer driver genes subject to amplifications and deletions, a peak-clustering method was performed to select genes frequently aberrant across multiple cancer types. First copy number peaks were defined across the largest-available set of copy number data (i.e data beyond what is included in the Integrative Analysis) within many cancer types. Next, the gene lists defined by the peaks were clustered in order to identify genes appearing in copy number peaks in multiple samples and multiple diseases. The parts of the method are described in more detail below.

An aberration may be classified as a “driver” aberration—or one that is considered potentially interesting according to one of the data type-specific Oncomine classification methods. Driver aberrations will be captured as events independently of other aberrations (non-driver aberrations are termed “any”). For example, a patient who has a “driver” mutation will be positive for two aberrations—a “driver” mutation, and an “any” mutation. Each of the measured data types has a set of rules for determining the driver events.

A set of continuous genomic regions subject to amplification or deletion were identified using the Oncomine MCR analysis by applying it to Oncomine's 10,249 clinical samples grouped into 25 cancers.

The patient null set for the peak definition was 10,249 clinical samples from Oncomine (See Table below). The gene null set for this data was the Oncomine DNA Copy Number platform, based on Ref Seq coordinates (hg18) provided by UCSC ref Gene build July 2009, and measures 18,796 genes.

Data for the minimal common region (MCR) analysis was sourced from Oncomine DNA copy number browser that contains >20,000 clinical specimens, xenografts and cell lines across diverse cancer types. MCR analysis identifies regions of recurrent copy number amplifications or deletions by analyzing the data at three levels—pan-cancer (across all cancer types), general cancer type (across cancer types), and intermediate cancer type or specific cancer sub-types. Briefly, the method first computes a common region (CR) defined as a contiguous genomic region that is amplified or deleted in 2 or more samples. The minimum thresholds for amplifications and deletions were set at log 2≥0.9 (3.7 copies or more) and log 2-0.9 (1 copy or less) respectively. Then the peak regions within these common regions are defined as—(i) one or more genes that are aberrant in the highest number of samples (n) and also those that are aberrant in one less than the highest number (n−1) and (ii) genes that are aberrant in 90% of the highest aberrant sample count.

Cluster Analysis to Identify Common Peaks Regions.

MCR analysis was performed. Peak regions identified by the MCR analysis were further filtered across the three analysis types (that is, pan-cancer, general cancer type, and specific cancer type analyses) using the criteria listed in table below. Note that only selected number (˜40) of intermediate or specific cancer types (also listed further below) were included.

Filtering criteria to identify highly amplified/deleted regions from MCR analysis:

Intermediate General cancer or specific Pan-cancer type cancer type Aberrant sample count ≥4 ≥4 ≥4 Maximum log2 copy ≥2 (8 or ≥2 (8 or ≥2 (8 or number- more copies) more copies) more copies) Amplifications Maximum log2 copy ≤−1 (1 or ≤−1 (1 or number- Deletions less copies) less copies) Median frequency ≥0.5% ≥0.5% ≥1.0% Intermediate or Include all Include all Selected ICTs specific cancer types (see Table 6)

Selected intermediate or specific cancer types included in the filtering criteria described above:

General Cancer Type Intermediate or specific cancer types Bladder Bladder Urothelial Carcinoma Brain and CNS Glioblastoma; Medulloblastoma; Neuroblastoma Breast N/A Cervical Cervical Adenocarcinoma; Cervical Squamous cell carcinoma Colorectal Cancer Colorectal Adenocarcinoma Esophageal Esophageal Adenocarcinoma; Esophageal squamous cell carcinoma Gastric Gastric Adenocarcinoma Head and Neck Head-Neck Squamous Cell Carcinoma; Thyroid gland follicular carcinoma; Thyroid Gland Papillary Carcinoma Kidney Clear Cell Renal Cell Carcinoma; Papillary Renal Cell Carcinoma Leukemia Acute Lymphoblastic Leukemia; Acute Myeloid Leukemia; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; Myelodysplastic Syndrome Liver Hepatocellular Carcinoma Lung Lung Adenocarcinoma; Small Cell Lung Carcinoma; Squamous Cancer Cell Lung Carcinoma Lymphoma Burkitt's Lymphoma; DLBCL; Follicular Lymphoma; Hodgkin's Lymphoma; Mantle Cell Lymphoma; Melanoma Cutaneous Melanoma; Multiple Myeloma Other Endometrial Endometrioid Adenocarcinoma Ovarian Ovarian Clear Cell Adenocarcinoma; Ovarian Serous Adenocarcinoma Pancreas Pancreatic Ductal Adenocarcinoma; Prostate Adenocarcinoma Sarcoma GIST

Next, to identify the most recurrent peak regions and genes across multiple cancer types we used Cytoscape 2.8.3 [Markiel et al. 2003; Smoot et al. 2001] to build network clusters. Briefly, the analysis compares every gene in a given peak region to genes in other peak regions and clusters peaks with at least one common gene. The most recurrent amplified or deleted gene(s) within each cluster was then considered as a potential candidate driver gene. The process is shown in FIG. 11 .

Identification and Creation of Clinically Relevant Cancer Subtypes. In order to provide subsets of patients for more focused analysis, several clinically relevant cancer subtypes were identified and curated using a combination of clinical phenomic, and categorical genomic data. The phenomic data was sourced from the TCGA Web Portal or the Supplementary Methods of the hand-curated publications.

The following rules were applied to define the Clinical Subsets:

Data Interpretation Rules for Inclusion in Cancer Type Clinical Subtype Data Source Subtype Invasive Breast Triple Negative TCGA Web Portal ERBB2 Status = “ERBB2 Negative” Carcinoma Estrogen Receptor Status = “Estrogen Receptor Negative” Progesterone Receptor Status = “Progesterone Receptor Negative” ER Positive Phenomic Estrogen Receptor Status = “Estrogen Receptor Positive” HER2 Positive Phenomic ERBB2 Status = “ERBB2 Positive” ER Positive and Phenomic Estrogen Receptor Status = “Estrogen HER2 Positive Receptor Positive” ERBB2 Status = “ERBB2 Positive” ER Positive and Phenomic Estrogen Receptor Status = “Estrogen HER2 Negative Receptor Positive” ERBB2 Status = “ERBB2 Negative” Gastric Diffuse Phenomic Cancer Type = “Diffuse Gastric Adenocarcinoma Intestinal Phenomic Adenocarcinoma” Cancer Type = “Gastric Intestinal Type Adenocarcinoma” Hyper-Mutator Oncomine NGS Patient Mutation Count ≥ 400 Head and Neck HPV Positive Phenomic Human Papillomavirus Infection Status = Squamous Cell “HPV Positive” Carcinoma HPV Negative Phenomic Human Papillomavirus Infection Status = “HPV Negative” Cervical HPV Positive Phenomic Human Papillomavirus Infection Status = Squamous Cell “HPV Positive” Carcinoma Lung EGFR Mutation Oncomine NGS Oncomine Mutation Classification = Adenocarcinoma Mutation “Hotspot” KRAS Mutation Oncomine NGS Oncomine Mutation Classification = (No ALK Fusion Mutation and “Hotspot” and No EGFR Fusion Mutation) ALK Fusion Oncomine NGS Have Oncomine Driver ALK fusions Fusion Triple Negative Oncomine NGS No EGFR Mutation Mutation and AND Fusion No KRAS Mutation AND No ALK Fusion Colon and Rectal KRAS Mutation Oncomine NGS Oncomine Mutation Classification = Adenocarcinoma Mutation “Hotspot” KRAS Mutation, Oncomine NGS Oncomine Mutation Classification = Stage 3 or 4 Mutation and “Hotspot” Phenomic AND Stage = “Stage III” OR “Stage IV” KRAS Wildtype Oncomine NGS Oncomine Mutation Classification = Mutation and “Hotspot” Fusion AND No KRAS Fusion Microsatellite Phenomic Microsatellite Status = “Microsatellite Stable Stable” Microsatellite Phenomic Microsatellite Status = “Microsatellite Instable Instable” Endometrial Microsatellite Phenomic Microsatellite Status = “Microsatellite Endometrioid Stable Stable” Carcinoma Microsatellite Phenomic Microsatellite Status = “Microsatellite Instable Instable”

Pathways.

Manually curated Compendia pathway definitions were used to summarize gene-level aberrations in the Integrative Analysis Browser. The pathways represent clinically relevant pathway modules, and several modules may cover a major biological pathway. A single gene may be present in one or more pathway definitions, but care was taken to eliminate largely redundant pathways, in which one module is a complete subset of another. There are 67 total pathways, ranging in size from 42 genes (e.g. MAPK pathway) to 2 genes (e.g. IGF1/IGF1R and several others).

Data Integration.

This section summarizes the data flow that integrates the primary data onto common patient and gene dimensions and constructs the Genetic Event Database (GEDB), which is comprised of all the aberrations which will be subject to Integrative Analyses. The process has 4 main steps: (1) Integrate primary data using universal gene and patient dimensions, (2) Call aberration events for each data type and define driver aberrations (3) Roll-up individual events to the gene and pathway level and integrate events, and (4) Construct the Genetic Event Database by defining patient status for each event.

Constructing and Mapping to the Gene and Patient Dimensions.

The varied data types included in the Integrative Analysis may have been measured on different experimental platforms and on sets of patients that are not perfectly overlapping. Therefore, care was taken to include all patients and genes measured while avoiding duplicate or conflicting entries.

For each data type, a gene and patient “dimension” was constructed, enumerating the genes and patients measured in the data. The dimension for each data type may be different, as indicated by the patient dimension overlap diagram below (numbers for illustration only), in this case, for Invasive Breast Carcinoma.

Gene and patient dimensions were gathered from each cancer and data type, and a non-redundant superset of all the patient and gene identifiers in the Integrative Analysis was constructed. The bars in the figure below represent blocks of patient identifiers (if sorted by said identifier) for patients measured for the certain aberration types.

Redundancy for patients was determined based on the unique patient identifier—currently the first 12 characters of the TCGA Tumor Sample Barcode (e.g., TCGA-AB-1234).

Redundancy for genes was determined based on the unique gene identifier—currently the Entrez Gene ID. The genes were also compared against the Oncomine gene set, and when a gene symbol conflict was found—one Entrez ID assigned two or more gene symbols—the gene symbol from Oncomine was used. Several (12) of the measured genes contained Entrez ID that have been discontinued and thus may not represent the most accurate gene model. The gene symbols for these genes were marked with the word “discontinued”.

Once constructed, the non-redundant gene and patient dimensions were indexed to provide a consistent internal identifier for each gene and patient in the dataset. All the unique patient and gene identifiers in the primary data were then mapped to the dimension patient and gene identifiers. Gene and patient metadata, such as gene symbols and patient clinical data, are thus always mapped through the respective dimensions, providing consistency in naming and annotation. The total number of unique genes and patients in the Integrative Analysis is as follows:

Genes 23,340 Patients 11,476

The patient dimension along with the dataset-specific mapping of the patients helps correctly identify fully wild-type patients—those who are measured for all aberration types but do not contain any aberrations.

A patient could thus be measured for any number of aberrations, and can only be aberrant for those events measured. The aberrations a patient is measured for determined the types of analyses that patient would be included in:

Patient Patient Patient Patient “X” “X” “Y” “Y” Measured Excluded Measured Excluded for: from: for: from: Clinical* Clinical ✓ DNA vs. RNA Mutations ✓ Associations, ✓ Correlation, Fusions ✓ Clinical ✓ Differential Copy Number ✓ Outcome ✓ Expression, Expression ✓ Associations Expressed Frequency *“Measured for: Clinical” indicates clinical metadata was present for patient.

Event Model.

Each genomic aberration from the mutation, fusion, and copy number data sets was identified as an aberration event—a term used to define an event of interest that will be subject to the various pre-defined Integrative Analyses. Each aberration is part of three broad levels of events—data type-specific events, gene-specific but data type independent events, and pathway-specific but gene or data type independent events. The latter two levels are considered “rolled-up” events.

The specific rules for aberration event definition as well as the “level” and “driver” schemes are described below.

Mutation Event Caller.

Oncomine Pan-Cancer Mutation Classification: A mutation is classified as a “Hotspot” if it is: Recurrent (occurs in 3 or more samples at the same amino acid position) ANDAnnotated with one of the following variant classifications: In-Frame insertion/deletion, Nonstop, Missense. A mutation is classified as “Deleterious” if it is: Not recurrent AND Annotated with one of the following variant classifications: Frame-Shift insertion/deletion, Nonsense. Recurrence is measured across all cancer types analyzed as part of the Oncomine NGS Mutation Browser.

Oncomine Pan-Cancer Gene Classification.

As part of the Oncomine NGS Mutation Browser pipeline, individual genes are classified into predicted functional classes, namely “Gain of Function” and “Loss of Function” to reflect their relative enrichment in potential activating or deleterious mutations. This classification is based on the combination of relative frequencies and the significance of the mutations observed in the gene assessed by a p-value. A “Gain of Function” gene will have a relatively high frequency of hotspot (recurrent in 3 or more samples) missense mutations and a low frequency of deleterious mutations, while a “Loss of Function” gene contains a large fraction of deleterious mutations. Pan-cancer gene classifications are based on the mutations observed across all cancer types.

Mutation Aberration Events.

For each patient gene mutation (as defined by the Mutation Data Filtering section), either one or two mutation events will be created, depending on whether the mutation is classified as a driver aberration. A driver mutation aberration is defined as a “Hotspot” mutation present in a “Gain of Function” gene, or a “Hotspot” or “Deleterious” mutation present in a “Loss of Function” gene. For driver mutations, both a driver event and an any event is created. For non-driver mutations, only an any event is created. Pan-Cancer mutation and gene classification was used for all analysis subsets; so, gene classifications may sometimes differ between Integrative Analysis and Oncomine NGS Mutation Browser.

The table below gives the description and examples of mutation events that could be created for each gene:

Event Example Driver Type Description of Event Aberration Name Status Gain of A “Hotspot” mutation and a EGFR Gain of driver Function “Gain of Function” gene Function Mutation Mutation classification Loss of A “Hotspot” or APC Loss of Function driver Function “Deleterious” mutation in a Mutation Mutation “Loss of Function” gene Any Gene Any mutation in a gene TTN <Any Gene any Mutation Mutation>

Fusion Event Caller.

Only Oncomine Priority fusions are included in the Integrative Analysis. Of the Priority Fusions, the driver fusions were defined as those labeled known oncogenes by the Mitelman database OR fusions that either did not have sufficient exon expression data and are recurrent, OR fusions that have exon expression data and a significant p-value for exon expression imbalance of the two gene partners (See Oncomine NGS Fusions Methods Documentation for details of exon imbalance classification). For each gene, an event will created for each unique observed 5′-3′ combination of the gene partners. For example, for PML-RARA balanced translocation both isoforms are observed and hence two fusion events will be called—for PML-RARA end RARA-PML respectively.

Event Example Driver Type Description of Event Aberration Name Status Fusion Driver fusion involving gene PML-RARA Fusion driver Any Any fusion involving gene FRS2-LYZ Fusion any Fusion

Copy Number Event Caller.

Each Amp/Del (see Calling Amp/Dels) that was called was defined as an any event for the aberrant gene. If the amp/del occurred in a gene that was part of a peak definition (see MCR Peak Generation) in a certain cancer type, a driver ampdel event was also created for that gene. The driver definition for copy number events is thus cancer type specific.

The following are the copy number aberration events that may be be called for a gene amplification or deletion:

Driver Event Type Description of Event Example Aberration Name Status In-Peak Gene An amplification in a gene EGFR In-Peak Gene driver Amplification observed in an Amplification Amplification Peak within the same cancer type In-Peak Gene A deletion in a gene observed CDKN1A In-Peak Gene Deletion driver Deletion in a Deletion Peak within the same cancer type Any Gene An amplification in a gene ERBB2 <Any Gene any Amplification Amplification> Any Gene A deletion in a gene FGFR <Any Gene Deletion> any Deletion

Genetic Event Roll-Up.

Both driver and any events are “rolled-up” to gene-level and pathway-level events to capture a data type-independent aberration statistics and associations. For example, it may be interesting to see the association of any aberrations in a tumor suppressor gene with clinical outcome, not just the association of the deleterious mutations.

A gene-level aberration event is created for each gene that has at least one aberration of any data type. A pathway-level aberration event is created for each pathway in which at least one of the component genes has an aberration of any data type. Driver and any aberrations are rolled-up independently into gene-level or pathway-level driver or any events. The diagram below shows the hierarchical relationships between the various aberration event types.

Patient Event Status.

A patient can be measured for any number of aberrations but can only be aberrant for those events measured. Patient status for each event-level aberration is thus recorded as aberrant, wild type, or not measured.

The patient dimension along with the data set-specific mapping of the patients helps correctly identify fully wild-type patients—those who are measured for all aberration types but don't contain any aberrations.

When gene-level and pathway-level events are defined, only the patients measured for all 3 genetic data types—mutations, fusion, and copy number—are marked as “aberrant” or “wild type” for the event. This assumption has the effect of potentially reducing the number of patients summarized for a gene or pathway-level aberrations as compared to the data type-specific event-level aberrations. A patient is considered aberrant for a gene-level event if the patient is aberrant for at least one of the event-level aberration types (Fusion, Mutation, Amplification, or Deletion). A patient is considered aberrant for a pathway-level event if the patient has an aberration in at least one gene that is part of the pathway definition. In each case, the patient must have been measured for all the event types.

In the case of the Gain of Function and Fusion, the aberration frequency is ˜50%. For the Driver Gene Aberration event the aberration frequency is also ˜50% but only half as many patients are included in the numerator and denominator of the frequency.

Analysis.

Once all the driver genetic events are constructed, a set of analyses is performed on each genetic event, calculating frequencies, associations, and relationships within cancer types, clinically relevant subtypes, and among cancer types (pan-cancer). The following are short descriptions of each analysis, including which data is used, and what constraints, if any, are put on the reported results: frequency, expressed frequency, co-occurrence and mutual exclusivity, clinical association analysis, etc.

Frequency.

Frequency is the occurrence of an aberration among the patients in which it was measured. Frequencies are calculated within cancer types, clinically relevant cancer subtypes, and pan-cancer. All events with at least one aberrant patient are reported.

Expressed Frequency.

Expressed frequency is the frequency at which the gene(s) is expressed among the event-positive patients. For each event, expression level of the gene(s) is used to ascertain the expressed frequencies. Frequencies are calculated within cancer types and clinically relevant cancer subtypes, but not pan-cancer.

Co-Occurrence and Mutual Exclusivity.

Co-occurrence and mutual exclusivity is calculated for each pair of events using a Fischer's Exact test. At least 2 patients positive for each event and 5 patients measured for the events in total are required for the calculation. Co-occurrence or exclusivity of two individual copy number deletion or amplification events is not calculated. Also, co-occurrence and mutual exclusivity is not calculated between pairs of events with “any” driver status (i.e. only drivers vs. drivers and drivers vs. any are compared). Associations are calculated within cancer types and clinically relevant cancer subtypes, but not pan-cancer.

Clinical Association Analysis.

Each driver event is tested for association against a set of available clinical subtypes. Each association is tested using a Fischer's exact test by comparing the occurrences of the genetic event in patients of one clinical subtype versus another. For example, a Loss of Function mutation may be tested for over-representation in Smokers versus Non-Smokers, or in Stage I versus Stage II lung cancer. A total of 136 subtype pairs are tested against each event, and the properties that define the subtypes are listed below (some properties may be disease-specific). At least 4 patients total, with at least 1 patient in each class are required to perform the test. Associations are calculated within cancer types, clinically relevant cancer subtypes, and pan-cancer.

Clinical Subtype Property Names:

-   -   Race/Ethnicity     -   Revised Smoking Status     -   ERBB2 Status     -   Estrogen Receptor Status     -   Progesterone Receptor Status     -   TCGA PAM50 Subtype     -   BRAF Mutation Status     -   Revised T Stage     -   Revised N Stage     -   Revised M Stage     -   Revised Stage     -   KRAS Mutation Status     -   EGFR Amplification Status     -   TCGA Subtype     -   Microsatellite Status     -   Human Papillomavirus Infection Status

Clinical Outcome Analysis.

Each event is tested for association with clinical outcome using the Logrank test. Only the set of patients with available clinical data are used for the calculation, so the number of patients included in the test may be less than the number of patients measured for the driver event. At least 4 patients aberrant for an event are required to perform the test. Survival time is presented in years, and individual alive/dead events are clearly marked on a Kaplan-Meier curve. Associations are calculated within cancer types and clinically relevant cancer subtypes, but not pan-cancer.

DNA-RNA Correlation Analysis.

For each gene, the RNA expression and DNA copy number values are tested for correlation among all patients within a disease who were measured for these data types using Pearson's correlation. Correlations are calculated within cancer types and clinically relevant cancer subtypes, but not pan-cancer.

Differential Expression Analysis.

For each event, each gene associated with the event was tested for differential expression in event-positive patients vs. event-negative patients using Student's T-Test. For events involving several genes—such as fusions—each gene was tested. Differential expression is calculated within cancer types and clinically relevant cancer subtypes, but not pan-cancer.

TABLE 15 Events associate with cancer prognosis Total Table 15 event p- q- No. no. of Subset type value value positive patients Cytoband Hepatocellular In-Peak 3.31E−02 9.93E−02 4 65 1q21.2 Carcinoma Gene Amplification Hepatocellular In-Peak 2.47E−02 9.89E−02 4 65 13q14.2 Carcinoma Gene Deletion Squamous Cell Lung Loss of 1.60E−02 9.59E−02 7 175 4q31.3 Carcinoma Function Mutation Squamous Cell Lung Loss of 3.14E−02 9.42E−02 7 175 9q34.3 Carcinoma Function Mutation Squamous Cell Lung Loss of 7.73E−03 9.28E−02 5 175 1p35.3 Carcinoma Function Mutation Clear Cell Renal Cell In-Peak 7.12E−03 9.25E−02 8 493 9p21 Carcinoma Gene Deletion Invasive Breast In-Peak 2.17E−03 9.13E−02 15 635 17q11.2 Carcinoma:ER Positive Gene Amplification Ovarian Serous In-Peak 1.00E−03 8.99E−02 10 557 19q13.1-q13.2 Cystadenocarcinoma Gene Amplification Clear Cell Renal Cell Loss of 2.44E−02 8.55E−02 14 293 3p21 Carcinoma Function Mutation Ovarian Serous In-Peak 5.45E−04 8.39E−02 89 557 19q12 Cystadenocarcinoma Gene Amplification Lung Adenocarcinoma In-Peak 6.80E−03 8.16E−02 4 320 1q12 Gene Amplification Lung Adenocarcinoma In-Peak 6.80E−03 8.16E−02 4 320 1q21.1 Gene Amplification Lung Adenocarcinoma In-Peak 8.57E−03 7.71E−02 9 320 12p12.1 Gene Amplification Invasive Breast In-Peak 8.10E−03 7.29E−02 5 88 8p12 Carcinoma:Triple Gene Negative Amplification Head and Neck In-Peak 1.02E−02 6.93E−02 8 316 5q35 Squamous Cell Gene Carcinoma Amplification Rectal In-Peak 2.08E−03 6.86E−02 4 145 16p13.3 Adenocarcinoma Gene Deletion Lung Adenocarcinoma In-Peak 9.37E−03 6.09E−02 5 320 3q25.1 Gene Deletion Hepatocellular In-Peak 3.03E−02 6.06E−02 4 65 8p21.2 Carcinoma Gene Deletion Ovarian Serous In-Peak 5.58E−04 6.02E−02 22 557 20q11 Cystadenocarcinoma Gene Amplification Rectal Loss of 2.69E−02 5.38E−02 11 28 5q21-q22 Adenocarcinoma:KRAS Function Wildtype Mutation Papillary Renal Cell In-Peak 2.68E−02 5.35E−02 6 100 17q21.1 Carcinoma Gene Amplification Acute Myeloid PML + RARA 1.26E−02 5.03E−02 15 169 17q and Leukemia Fusion 15q Rectal In-Peak 4.55E−02 4.96E−02 10 27 20q Adenocarcinoma:KRAS Gene Wildtype Amplification Gastric Loss of 4.09E−03 4.50E−02 4 131 6p21 Adenocarcinoma Function Mutation Endometrial In-Peak 1.36E−02 4.43E−02 6 446 16Q24 Endometrioid Gene Adenocarcinoma Deletion Lung Adenocarcinoma In-Peak 3.15E−03 4.09E−02 8 320 19q13.4 Gene Deletion Head and Neck In-Peak 1.45E−03 3.82E−02 6 316 20p12 Squamous Cell Gene Carcinoma Amplification Lung In-Peak 8.80E−04 3.78E−02 6 174 7q31 Adenocarcinoma:Triple Gene Negative Amplification Endometrial In-Peak 1.09E−02 3.77E−02 4 446 3Q26 Endometrioid Gene Adenocarcinoma Deletion Cutaneous Melanoma Loss of 3.74E−03 3.74E−02 16 148 17q11 Function Mutation Acute Myeloid CBFB + MYH11 1.83E−02 3.67E−02 11 169 16Q22 and Leukemia Fusion 16P13.11 Head and Neck In-Peak 6.01E−04 2.86E−02 5 316 7p12 Squamous Cell Gene Carcinoma Amplification Lung Adenocarcinoma In-Peak 1.28E−03 2.76E−02 7 320 7q31 Gene Amplification Head and Neck In-Peak 3.00E−03 2.31E−02 80 316 11q13 Squamous Cell Gene Carcinoma Amplification Gastric In-Peak 1.89E−04 2.14E−02 4 172 18q11 Adenocarcinoma Gene Amplification Invasive Breast In-Peak 2.27E−03 1.82E−02 11 863 10q23.31, Carcinoma Gene q23.2 Deletion Head and Neck In-Peak 1.89E−03 1.55E−02 6 316 2q32 Squamous Cell Gene Carcinoma Amplification Colon In-Peak 2.27E−04 1.48E−02 4 412 3Q26 Adenocarcinoma Gene Deletion Gastric Loss of 5.32E−04 1.22E−02 4 32 2q31 Adenocarcinoma:Hyper- Function Mutator Mutation Glioblastoma Loss of 1.23E−03 1.11E−02 6 276 Xq25 Function Mutation Head and Neck Gain of 2.61E−03 1.04E−02 13 304 2q31 Squamous Cell Function Carcinoma Mutation Endometrial In-Peak 9.20E−04 1.03E−02 7 446 1q21 Endometrioid Gene Adenocarcinoma Amplification Endometrial In-Peak 2.47E−03 9.17E−03 7 446 16p13 Endometrioid Gene Adenocarcinoma Deletion Hepatocellular In-Peak 2.57E−03 8.89E−03 4 65 1q21.3 Carcinoma Gene Amplification Clear Cell Renal Cell In-Peak 3.16E−04 8.23E−03 8 493 9p21 Carcinoma Gene Deletion Glioblastoma Gain of 2.72E−03 8.15E−03 14 276 2q33 Function Mutation Lung In-Peak 2.56E−03 5.98E−03 4 78 12p12.1 Adenocarcinoma:KRAS Gene Mutation (No ALK Amplification Fusion and No EGFR Mutation) Endometrial In-Peak 4.40E−04 5.55E−03 6 446 1q22 Endometrioid Gene Adenocarcinoma Amplification Colon Gain of 4.97E−03 4.97E−03 17 53 3q26 Adenocarcinoma:KRAS Function Mutation Mutation Head and Neck Loss of 1.79E−04 3.95E−03 161 304 17p13 Squamous Cell Function Carcinoma Mutation Head and Neck In-Peak 6.41E−05 3.81E−03 4 316 22q11 Squamous Cell Gene Carcinoma Amplification Gastric Loss of 2.14E−04 3.53E−03 5 131 17q22 Adenocarcinoma Function Mutation Lower Grade Glioma Loss of 3.00E−04 2.70E−03 5 166 17q11.2 Function Mutation Lung Gain of 5.06E−04 2.53E−03 11 175 3q26.3 Adenocarcinoma:Triple Function Negative Mutation Lung Adenocarcinoma Loss of 5.24E−05 9.96E−04 4 283 5q21-q22 Function Mutation Bladder Urothelial In-Peak 8.34E−05 9.31E−04 5 125 5p15.33 Carcinoma Gene Amplification Endometrial In-Peak 1.12E−04 8.32E−04 9 446 10q23 Endometrioid Gene Adenocarcinoma Deletion Lower Grade Glioma In-Peak 5.69E−04 6.74E−04 5 206 1q32.1 Gene Amplification Ovarian Serous In-Peak 1.05E−06 6.28E−04 7 557 9q22 Cystadenocarcinoma Gene Deletion Endometrial In-Peak 8.93E−06 3.01E−04 25 446 8q24 Endometrioid Gene Adenocarcinoma Amplification Acute Myeloid Loss of 2.35E−05 9.42E−05 12 184 17P Leukemia Function Mutation Colon In-Peak 3.93E−06 6.24E−05 7 412 12p13 Adenocarcinoma Gene Amplification Gastric Loss of 8.74E−07 2.88E−05 4 131 2q31 Adenocarcinoma Function Mutation Lower Grade Glioma Gain of 9.38E−08 2.81E−07 130 166 2q33.3 Function Mutation Lower Grade Glioma In-Peak 1.31E−08 3.48E−08 14 206 7p11.2 Gene Amplification Lower Grade Glioma In-Peak 1.48E−10 1.18E−09 5 206 12q14.1 Gene Amplification Lower Grade Glioma Gain of 1.09E−10 6.56E−10 6 166 7p12 Function Mutation Lung Adenocarcinoma In-Peak 1.30E−12 4.66E−11 4 320 12p11 Gene Amplification Lower Grade Glioma In-Peak 4.57E−12 6.85E−12 21 206 9p21 Gene Deletion Endometrial In-Peak 2.00E−15 1.01E−13 4 446 17q21 Endometrioid Gene Adenocarcinoma Amplification Astrocytoma Loss of 3.88E−03 34 59 17p13.1 Function Mutation Astrocytoma Loss of 8.15E−03 22 59 Xq21.1 Function Mutation Breast Carcinoma In-Peak 8.14E−03 4 36 8p23.2 Gene Deletion Colorectal In-Peak 5.71E−02 12 407 8q24.3 Adenocarcinoma Gene Amplification Colorectal In-Peak 9.18E−02 17 407 13q34 Adenocarcinoma Gene Amplification Colorectal Mucinous Gain of 8.10E−03 8 32 3q26.3 Adenocarcinoma Function Mutation Cutaneous Melanoma In-Peak 2.60E−06 6 231 8q22.3 Gene Amplification Cutaneous Melanoma In-Peak 1.54E−04 7 231 8q24.3 Gene Amplification Cutaneous Melanoma In-Peak 7.21E−03 8 231 8q21 Gene Amplification Cutaneous Melanoma In-Peak 7.59E−03 6 231 11q13.3 Gene Amplification Cutaneous Melanoma In-Peak 1.82E−02 4 231 1q44 Gene Amplification Cutaneous Melanoma In-Peak 9.36E−02 6 231 1q21.3 Gene Amplification Ductal Breast In-Peak 2.77E−03 4 665 3q29 Carcinoma Gene Amplification Ductal Breast In-Peak 2.28E−02 7 665 6q23.3 Carcinoma Gene Amplification Ductal Breast In-Peak 2.64E−02 8 665 3q26.3 Carcinoma Gene Amplification Ductal Breast In-Peak 7.92E−06 6 263 1q21.3 Carcinoma:ER Positive Gene and HER2 Negative Amplification Ductal Breast In-Peak 4.02E−02 7 263 1q32 Carcinoma:ER Positive Gene and HER2 Negative Amplification Ductal Breast In-Peak 4.35E−02 4 263 8p11.2 Carcinoma:ER Positive Gene and HER2 Negative Deletion Ductal Breast In-Peak 7.48E−02 4 84 9q22 Carcinoma:ER Positive Gene and HER2 Positive Deletion Ductal Breast In-Peak 4.47E−02 4 116 15q13.1 Carcinoma:HER2 Gene Positive Deletion Ductal Breast In-Peak 5.17E−02 4 116 9p21 Carcinoma:HER2 Gene Positive Deletion Ductal Breast In-Peak 2.58E−02 5 75 1q23.3 Carcinoma:Triple Gene Negative Amplification Ductal Breast In-Peak 7.21E−02 8 75 1q21 Carcinoma:Triple Gene Negative Amplification Endometrial Loss of 5.55E−02 19 113 5q13.1 Endometrioid Function Adenocarcinoma:Microsatellite Mutation Stable Endometrial Serous In-Peak 6.37E−04 4 52 19p13.2 Adenocarcinoma Gene Amplification Gastric In-Peak 9.05E−02 8 106 9p21 Adenocarcinoma:Hyper- Gene Mutator Deletion Glioblastoma In-Peak 2.58E−02 300 565 9p21 Gene Deletion Glioblastoma In-Peak 8.80E−02 189 565 7p11.2 Gene Amplification Lung Adenocarcinoma Fusion 5.79E−02 7 343 17q23.1 Lung Loss of 1.31E−03 4 99 7q36.1 Adenocarcinoma:Triple Function Negative Mutation Oligoastrocytoma Loss of 1.97E−02 38 53 17p13.1 Function Mutation Oligodendroglioma Loss of 5.90E−02 6 89 9q34.3 Function Mutation Oligodendroglioma Loss of 6.62E−02 15 89 1p31.1 Function Mutation Ovarian Serous In-Peak 1.15E−02 17 562 19q13.1 Adenocarcinoma Gene Amplification Ovarian Serous In-Peak 6.59E−02 17 562 20q11.2-13.2 Adenocarcinoma Gene Amplification Ovarian Serous In-Peak 7.86E−02 7 562 17p13.1 Adenocarcinoma Gene Deletion Ovarian Serous In-Peak 8.43E−02 53 562 19q12 Adenocarcinoma Gene Amplification Squamous Cell Lung In-Peak 7.93E−02 63 320 3q26.2 Carcinoma Gene Amplification Table 15 Genes (Entrez Druggable KM Subset ID) genes Evidence Hepatocellular ADAMTSL4 MCL1 Poor Carcinoma (54507), MCL1 prognosis (4170) Hepatocellular LPAR6 (10161) N Poor Carcinoma prognosis Squamous Cell Lung FBXW7 (55294) N Poor Carcinoma prognosis Squamous Cell Lung NOTCH1 NOTCH1 Poor Carcinoma (4851) prognosis Squamous Cell Lung ARID1A (8289) N Poor Carcinoma prognosis Clear Cell Renal Cell CDKN2B No Poor Carcinoma (1030) prognosis Invasive Breast TIAF1 (9220), N Poor Carcinoma:ER Positive MYO18A prognosis (399687), CRYBA1 (1411) Ovarian Serous PSG2 (5670), CEACAM1 Poor Cystadenocarcinoma PSG5 (5673), (Preclinical) prognosis CEACAM1 (634), CEACAM8 (1088), CXCL17 (284340), RABAC1 (10567), ATP1A3 (478) Clear Cell Renal Cell BAP1 (8314) No Poor Carcinoma prognosis Ovarian Serous C19orf2 (8725) N Poor Cystadenocarcinoma prognosis Lung Adenocarcinoma CHD1L (1105) N Poor prognosis Lung Adenocarcinoma FMO5 (2330), N Poor PRKAB2 (5565) prognosis Lung Adenocarcinoma KRAS (3845), KRAS Poor CASC1 (55259), (Preclinical) prognosis LYRM5 (144363), LRMP (4033) Invasive Breast BRF2 (55290), N Poor Carcinoma:Triple ERLIN2 prognosis Negative (11160), GPR124 (25960), PROSC (11212), RAB11FIP1 (80223), ZNF703 (80139) Head and Neck THOC3 (84321) No Poor Squamous Cell prognosis Carcinoma Rectal A2BP1 (54715) N Poor Adenocarcinoma prognosis Lung Adenocarcinoma AADAC (13) N Poor prognosis Hepatocellular GNRH1 (2796) GNRH1 Poor Carcinoma prognosis Ovarian Serous ID1 (3397), ID1 Poor Cystadenocarcinoma BCL2L1 (598), (Preclinical), prognosis COX4I2 BCL2L1 (84701) Rectal APC (324) N Poor Adenocarcinoma:KRAS prognosis Wildtype Papillary Renal Cell CCL3L3 N Poor Carcinoma (414062), prognosis CCL3L1 (6349) Acute Myeloid RARA (5914), Y Good Leukemia PML (5371) prognosis Rectal ACOT8 (10005), ADA, Poor Adenocarcinoma:KRAS ADA (100), CD40 prognosis Wildtype C20orf111 (958), (51526), MMP9, C20orf123 PI3 (128506), C20orf165 (128497), CD40 (958), CDH22 (64405), CTSA (5476), DBNDD2 (55861), DNTTIP1 (116092), ELMO2 (63916), FITM2 (128486), GDAP1L1 (78997), GTSF1L (149699), HNF4A (3172), IFT52 (51098), JPH2 (57158), KCNK15 (60598), KCNS1 (3787), L3MBTL (26013), MATN4 (8785), MMP9 (4318), MYBL2 (4605), NCOA5 (57727), NEURL2 (140825), PABPC1L (80336), PCIF1 (63935), PI3 (5266), PIGT (51604), PKIG (11142), PLAGL2 (5326), PLTP(5360), POFUT1 (23509), R3HDML (140902), RBPJL (11317), RIMS4 (140730), SDC4 (6385), SEMG1(6406) SEMG2 (6407), SERINC3 (10955), SFRS6 (6431), SGK2 (10110), SLC12A5 (57468), SLC13A3 (64849), SLC35C2 (51006), SLPI (6590), SNAI1 (6615), SNX21 (90203), SPINLW1 (57119), SPINT3 (10816), SPINT4 (391253), STK4 (6789), SYS1 (90196), TM9SF4 (9777), TNNC2 (7125), TOMM34 (10953), TOX2 (84969), TP53RK (112858), TP53TG5 (27296), TTPAL (79183), UBE2C (11065), WFDC10A (140832), WFDC10B (280664), WFDC11 (259239), WFDC12 (128488), WFDC13 (164237), WFDC2 (10406), WFDC3 (140686), WFDC5 (149708), WFDC6 (140870), WFDC8 (90199), WFDC9 (259240), WISP2 (8839), YWHAB (7529), ZNF334 (55713), ZNF335 (63925), ZSWIM1 (90204), ZSWIM3 (140831) Gastric HLA-B (3106) Yes Poor Adenocarcinoma prognosis Endometrial SLC7A5 (8140), SLC7A5 Poor Endometrioid CTU2 (66965), (preclinical) prognosis Adenocarcinoma FAM38A (9780), CDT1 (81620), APRT (353), GALNS (2588) Lung Adenocarcinoma KIR2DS4 N Poor (3809) prognosis Head and Neck C20orf94 JAG1 Poor Squamous Cell (128710), JAG1 (Preclinical) prognosis Carcinoma (182), MKKS (8195), SNAP25 (6616) Lung MET (4233), MET Poor Adenocarcinoma:Triple CAPZA2 (830) prognosis Negative Endometrial APOD (347) No Poor Endometrioid prognosis Adenocarcinoma Cutaneous Melanoma NF1 (4763) No Poor prognosis Acute Myeloid CBFB (865), N Good Leukemia MYH11 (4629) prognosis Head and Neck ABCA13 No Poor Squamous Cell (154664), prognosis Carcinoma C7orf57 (136288), C7orf65 (401335), C7orf69 (80099), C7orf72 (100130988), DDC (1644), FIGNL1 (63979), GRB10 (2887), HUS1 (3364), IKZF1 (10320), PKD1L1 (168507), SUN3 (256979), TNS3 (64759), UPP1 (7378), VWC2 (375567), ZPBP (11055) Lung Adenocarcinoma MET (4233), MET Poor CAPZA2 (830) prognosis Head and Neck FADD (8772), No Poor Squamous Cell PPFIA1 (8500), prognosis Carcinoma ANO1 (55107), CTTN (2017) Gastric GATA6 (2627) No Poor Adenocarcinoma prognosis Invasive Breast ATAD1 (84896), N Poor Carcinoma KILLIN prognosis (100144748) Head and Neck GLS (2744), No Poor Squamous Cell MYO1B (4430), prognosis Carcinoma NAB1(4664), STAT1 (6772), STAT4 (6775), TMEM194B (100131211) Colon APOD (347) No Poor Adenocarcinoma prognosis Gastric HOXD8 (3234) No Poor Adenocarcinoma:Hyper- prognosis Mutator Glioblastoma STAG2 (10735) No Poor prognosis Head and Neck NFE2L2 (4780) NO Poor Squamous Cell prognosis Carcinoma Endometrial SSR2 (6746), No Poor Endometrioid ARHGEF2 prognosis Adenocarcinoma (9181), UBQLN4 (56893) Endometrial LOC339047 No Poor Endometrioid (339047) prognosis Adenocarcinoma Hepatocellular DCST1 ADAM15, Poor Carcinoma (149095), MUC1 prognosis ADAM15 (8751), EFNA4 (1945), EFNA3 (1944), EFNA1 (1942), RAG1AP1 (55974), DPM3 (54344), KRTCAP2 (200185), TRIM46 (80128), MUC1 (4582), THBS3 (7059), MTX1 (4580), GBA (2629) Clear Cell Renal Cell CDKN2A Yes Poor Carcinoma (1029) prognosis Glioblastoma IDH1 (3417) preclinical Good prognosis Lung LYRM5 KRAS Poor Adenocarcinoma:KRAS (144363), (Preclinical) prognosis Mutation (No ALK KRAS (3845), Fusion and No EGFR CASC1 (55259) Mutation) Endometrial ROBLD3 No Poor Endometrioid (28956), RAB25 prognosis Adenocarcinoma (57111), MEX3A (92312) Colon PIK3CA (5290) Yes Poor Adenocarcinoma:KRAS prognosis Mutation Head and Neck TP53 (7157) TP53 Poor Squamous Cell prognosis Carcinoma Head and Neck CRKL (1399), No Poor Squamous Cell PI4KA prognosis Carcinoma (5297), SERPIND1 (3053), SNAP29 (9342) Gastric RNF43 (54894) No Poor Adenocarcinoma prognosis Lower Grade Glioma NF1 (4763) N Poor prognosis Lung PIK3CA (5290) Y Poor Adenocarcinoma:Triple prognosis Negative Lung Adenocarcinoma APC (324) N Poor prognosis Bladder Urothelial PLEKHG4B AHRR, Poor Carcinoma (153478), TERT prognosis LRRC14B (389257), CCDC12 (151903), SDHA (6389), PDCD6 (10016), AHRR (57491), C5orf55 (116349), EXOC3 (11336), SLC9A3 (6550), CEP72 (55722), TPPP (11076), BRD9 (65980), TRIP13 (9319), NKD2 (85409), SLC12A7 (10723), SLC6A19 (340024), SLC6A18 (348932), TERT (7015), CLPTM1L (81037), SLC6A3 (6531), LPCAT1 (79888), MRPL36 (64979), NDUFS6 (4726) Endometrial PTEN (5728), Yes Poor Endometrioid ANKRD22 prognosis Adenocarcinoma (118932), STAMBPL1 (57559), ACTA2 (59), FAS (355), ATAD1 (84896), KILLIN (100144748), RNLS (55328) Lower Grade Glioma C1orf157 MDM4 Poor (284573), (Preclinical) prognosis ETNK2 (55224), GOLT1A (127845), KISS1 (3814), LAX1 (54900), LRRN2 (10446), MDM4 (4194), PIK3C2B (5287), PLEKHA6 (22874), PPP1R15B (84919), REN (5972), SNRPE (6635), SOX13 (9580), ZC3H11A (9877) Ovarian Serous FAM75C1 N Poor Cystadenocarcinoma (441452) prognosis Endometrial MYC (4609), No Poor Endometrioid TAF2 (6873), prognosis Adenocarcinoma DSCC1 (79075), DEPDC6 (64798) Acute Myeloid TP53 (7157) Y Poor Leukemia prognosis Colon CCND2 (894), No Poor Adenocarcinoma TULP3 (7289), prognosis TEAD4 (7004), TSPAN9 (10867), PRMT (563418), EFCAB4B (84766), PARP11 (57097), C12orf5 (57103), FGF23 (8074), FGF6 (2251), FKBP4 (2288), ITFG2 (55846), NRIP2 (83714), FOXM1 (2305) Gastric HOXD8 (3234) No Poor Adenocarcinoma prognosis Lower Grade Glioma IDH1 (3417) IDH1 Good (Preclinical) prognosis Lower Grade Glioma EGFR (1956), EGFR poor SEC61G prognosis (23480) Lower Grade Glioma CDK4 (1019), CDK4 Poor CYP27B1 prognosis (1594), MARCH9 (92979), TSPAN31 (6302), AGAP2 (116986), AVIL (10677), CTDSP2 (10106), FAM119B (25895), METTL1 4234), OS9 (10956), TSFM (10102) Lower Grade Glioma EGFR (1956) EGFR Poor prognosis Lung Adenocarcinoma LOC100133893 N Poor (100133893), prognosis MRPS3 (604885), REP15 (387849) Lower Grade Glioma CDKN2A CDKN2A Poor (1029), (1029) prognosis CDKN2B (1030), MTAP (4507) Endometrial CCL3L3 No Poor Endometrioid (414062), prognosis Adenocarcinoma CCL3L1 (6349) Astrocytoma TP53 (7157) TP53 favorable outcome Astrocytoma ATRX (546) no favorable outcome Breast Carcinoma CSMD1 (64478) no poor outcome Colorectal PARP10 PTK2 poor Adenocarcinoma (84875), outcome MAPK15 (225689), PTK2 (5747), KHDRBS3 (10656) Colorectal FAM70B no poor Adenocarcinoma (348013) outcome Colorectal Mucinous PIK3CA (5290) PIK3CA poor Adenocarcinoma outcome Cutaneous Melanoma ODF1 (4956) no poor outcome Cutaneous Melanoma PARP10 PTK2 poor (84875), outcome MAPK15 (225689), PTK2 (5747), KHDRBS3 (10656) Cutaneous Melanoma HEY1 (23462) no poor outcome Cutaneous Melanoma FADD (8772), CCND1 poor CCND1 (595), outcome ORAOV1 (220064), FGF19 (9965) Cutaneous Melanoma OR2T27 no poor (403239) outcome Cutaneous Melanoma LCE1E no poor (353135) outcome Ductal Breast OSTalpha no poor Carcinoma (200931) outcome Ductal Breast AHI1 (54806) no poor Carcinoma outcome Ductal Breast PIK3CA (5290), PIK3CA poor Carcinoma SOX2 (6657), outcome ATP11B (23200) Ductal Breast ADAMTSL4 MCL1 poor Carcinoma:ER Positive (54507), MCL1 outcome and HER2 Negative (4170), ENSA (2029) Ductal Breast MDM4 (4194) MDM4 poor Carcinoma:ER Positive (pre- outcome and HER2 Negative clinical) Ductal Breast FKSG2 (59347) no poor Carcinoma:ER Positive outcome and HER2 Negative Ductal Breast FAM75C1 no poor Carcinoma:ER Positive (441452) outcome and HER2 Positive Ductal Breast CHRFAM7A no poor Carcinoma:HER2 (89832) outcome Positive Ductal Breast CDKN2B CDKN2B poor Carcinoma:HER2 (1030) (pre- outcome Positive clinical) Ductal Breast APOA2 (336), no poor Carcinoma:Triple SDHC (6391), outcome Negative FCGR2B (2213) Ductal Breast ACP6 (51205), MCL1 poor Carcinoma:Triple ECM1 (1893), outcome Negative ADAMTSL4 (54507), MCL1 (4170), ENSA (2029) Endometrial PIK3R1 (5295) no poor Endometrioid outcome Adenocarcinoma:Microsatellite Stable Endometrial Serous DNMT1 (1786) DNMT1 poor Adenocarcinoma outcome Gastric CDKN2A CDKN2A, poor Adenocarcinoma:Hyper- (1029), CDKN2B outcome Mutator CDKN2B (pre- (1030) clinical) Glioblastoma CDKN2A CDKN2A, poor (1029), CDKN2B outcome CDKN2B (pre- (1030) clinical) Glioblastoma SEC61G no poor (23480) outcome Lung Adenocarcinoma RPS6KB1 RPS6KB1 poor (6198), VMP1 outcome (81671) Lung MLL3 (58508) no poor Adenocarcinoma:Triple outcome Negative Oligoastrocytoma TP53 (7157) TP53 favorable outcome Oligodendroglioma NOTCH1 NOTCH1 poor (4851) outcome Oligodendroglioma FUBP1 (8880) no poor outcome Ovarian Serous FCGBP (8857), PAK4 poor Adenocarcinoma PAK4 (10298) (pre- outcome clinical) Ovarian Serous ZNF217 (7764), no poor Adenocarcinoma MYLK2 outcome (85366), KIF3B (9371) Ovarian Serous ATP1B2 (482) no poor Adenocarcinoma outcome Ovarian Serous CCNE1 (898) CCNE1 poor Adenocarcinoma outcome Squamous Cell Lung MECOM (2122) no favorable Carcinoma outcome

Example 5: Additional Fusion Methods

Clinical Data Sources.

All RNASeq data for gene fusion analysis was obtained from the Cancer Genomics Hub (CGHub), the current repository for TOGA genomic data—cghub.ucsc.edu.

Cell Line Data Sources.

All COLE RNASeq data for gene fusion analysis was obtained from the Cancer Genomics Hub (CGHub), the current repository for COLE NGS data—cghub.ucsc.edu.

BAM to FASTQ Conversion.

The input to the fusion callers consists of RNASeq reads in FASTQ format, which required conversion of the BAM file provided by TOGA to one or two FASTQ files for single or paired end data (respectively).

BAM files varied in provenance and processing, and many required special handling. For example, older BAM files provided by UNC were aligned using BWA (Burrows-Wheeler Aligner), while newer BAMs contained reads aligned by MapSplice. TOGA recently updated the RNASeq pipeline to support alternative gene expression reporting. (The former pipeline relied on the RPKM measurements for gene expression, while the latter uses RSEM.) These different RNASeq analysis pipelines are referred to by UNC as V1 and V2 respectively (wiki.nci.nih.gov/display/TCGA/RNASeq+Version+2). We used the following BAM prioritization pipeline to select a single “primary BAM” when both formats are available for the same TOGA sample: 1) V2 BAMs were chosen over V1 BAMs and 2) BAMs with newer upload dates were selected when multiple files for the same case were present.

The custom SamToFastq converter described above was used to generate FASTQ files from a TOGA BAM file.

There were 2 cancer types (COADREAD and UCEC) only available as single-end RNASeq data. For single-end BAM file conversion, the program BamTools (github.com/pezmaster31/bamtools) was used to generate FASTQ files.

With the goal of supporting both single and paired-end data, we processed all single-end data using TopHat and all paired-end data using deFuse.

Broadly, our analysis pipeline consists of 5 main steps:

Pre-process the raw data to obtain FASTQ files

Run fusion callers

Filter breakpoints to gene regions of interest

Annotate the breakpoints with the Oncomine transcript set

Summarize and prioritize potentially interesting novel fusions

Steps 1 and 2 were executed in parallel for all samples on a high-performance cloud computing cluster. The filtering and annotation was conducted on the aggregated data as a post-processing step, to enable exploratory analyses of effects of various filters and annotation schemes. After finalizing filtering criteria to minimize false positive fusions (Step 5), the list of Oncomine Prioritized Fusions is validated with RNASeq Exon Expression data.

TopHat.

TopHat-Fusion was obtained from the authors (tophat.cbcb.umd.edu). Software and reference data dependencies were configured as specified by the TopHat documentation:

Software:

TopHat: 2.0.4, includes TopHat-Fusion Post (release Apr. 9, 2012)

bowtie: 0.12.8 (release May 6, 2012)

samtools: 0.1.18 (release Sep. 2, 2011)

blast (2.2.26) (release Mar. 3, 2012)

blast+ (2.2.26) (release Oct. 21, 2011)

Reference and Annotation:

Reference Genome: UCSC hq19 (downloaded May 2012)

Gene Models: refGene, ensGene (downloaded May 2012)

BLAST DB: nt, human, other (downloaded May 2012)

Parameters:

We ran TopHat with largely default parameters on single and paired-end TOGA Illumina data as specified in the TopHat documentation. The following is a list of parameters used.

TABLE 25 TopHat Parameter Value Used --fusion-search Flag --keep-fasta-order Flag --no-coverage-search Flag --mate-inner-dist 0 --mate-std-dev 80 --min-anchor-length 8 --splice-mismatches 0 --min-intron-length 70 --max-intron-length 500,000 --max-insertion-length 3 --max-deletion-length 3 --num-threads 4 --max-multihits 20 --transcriptome-mismatches 2 --genome-read-mismatches 2 --read-mismatches 2 --segment-mismatches 2 --segment-length 25 --fusion-min-dist 100,000 --fusion-anchor-length 13 --fusion-read-mismatches 2 --fusion-multireads 2 --fusion-multipairs 2 --fusion-ignore-chromosomes chrM

The—mate-inner-dist and—mate-std-dev parameters have no default values. The first parameter specifies an expected insert size for the RNASeq paired-end reads, while the second parameters specifies the expected standard deviation of that value. The values of 0 and 80 are recommended by TopHat authors for most data sets.

TABLE 26 TopHat-Fusion Post Parameter Value Used Explanation of Values --num-fusion-reads 3 Recommended value --num-fusion-pairs 0 Set to 0 to not penalize low- --num-fusion-both 0 evidence, but potentially important fusions

TopHat-Fusion was executed on one sample at a time, immediately followed by TopHat-Fusion Post. We retained both, unfiltered TopHat-Fusion output and filtered TopHat-Fusion Post output, to enable deeper analyses.

deFuse.

deFuse was obtained from the authors: defuse.sf.net. Software and reference data dependencies were configured as specified by the deFuse documentation:

Software:

deFuse: 0.5.0 (released Apr. 7, 2012)

bowtie: 0.12.8 (release May 6, 2012)

R 2.15.0 (release Mar. 30, 2012)

blat, faToTwoBit (obtained on May 1, 2012)

Reference and Annotation

Reference Genome: Ensembl GRCh37.62 fa (downloaded May 2012)

Gene Models: Ensembl gtf (downloaded May 2012)

Genomic Data:

UCSC EST fasta, EST alignments, and repeats (downloaded May 2012)

NCBI UniGene (downloaded May 2012)

Parameters:

We ran deFuse with default parameters, as specified in the deFuse program documentation.

TABLE 27 deFuse Parameter Value Used -bowtie_quals phred33-quals -max_insert_size 500 -discord_read_trim 50 -clustering_precision 0.95 -span_count_threshold 5 -split_count_threshold 3 -percent_identity_threshold 0.90 -max_dist_pos 600 -num_dist_genes 500 -split_min_anchor 4 -max_concordant_ratio 0.1 -splice_bias 10 -denovo_assembly No -probability_threshold 0.5 -covariance_sampling_density 0.01 -reads_per_job 1,000,000 -regions_per_job 20 -p 4

deFuse was executed on one sample at a time. We kept both the filtered and unfiltered results of deFuse output to enable deeper analysis.

Integration.

We integrated the “Level I” data—the output from TopHat-Fusion Post's potential_fusion.txt file and the output from deFuse's results.classify.tsv file. deFuse reports many more potential calls at this level than TopHat, and thus may also report more false-positive predictions. The Level I data was chosen to strike a balance between utilizing the caller's built-in filtering and allowing through enough results to identify potentially real fusions with somewhat weaker evidence.

As each caller provided a different level of annotation and supporting evidence for the fusion calls, the breakpoints of the predicted fusions from both callers were extracted and integrated into a common format for filtering and annotation. The integration steps consisted of converting the reported breakpoints to ones-based genomic coordinate system, and consolidation into a common file format.

Breakpoint Filtering.

The predicted fusions from the “Level I” output of the callers were filtered to only retain those calls where each breakpoint was either in the 5′UTR or CDS region of a RefSeq transcript (refGene circa Jul. 18, 2012, obtained from UCSC). This was done to enrich the predicted fusions for those containing functional gene regions, filtering out, for example, fusions calls where the 3′UTR of one gene is predicted to be fused to a 3′UTR of another gene. Although at the genomic DNA level breakpoints may occur in introns, in RNASeq data such breakpoints would be observed at the nearest exon-intron boundary. Therefore, breakpoints predicted to occur in intronic sequences were also excluded.

Breakpoint Annotation.

After excluding fusions outside of the 5′UTR or CDS region of a RefSeq transcript, the annotation from the RefSeq transcripts was transferred to the remaining breakpoints with some predictions annotated against multiple Entrez IDs.

For each pair of breakpoints, only one transcript per Entrez ID was retained. In case of multiple transcripts, the transcript with the shortest transcript accession was chosen; further ties were broken by sorting the accessions alphanumerically and retaining the first accession. This scheme ensured consistency in annotating breakpoints at the same location. However, predicted breakpoints at different locations for the same gene partners may still result in multiple transcripts representing a pair of genes—possible evidence of alternative transcripts.

Basic annotation coming from the callers themselves was discarded, as it was based on the default annotation source of each respective caller. However, certain output fields from both TopHat and deFuse were retained to help prioritize the predicted fusions. Additionally, certain annotation properties that weren't explicitly reported by the callers were inferred from other caller properties.

Inferred Properties.

Supporting and Spanning read counts were obtained from each caller and summarized in two columns—Reads Span and Reads Span Support. The latter column is a sum of reads spanning the fusion and those supporting the fusion (not to be confused with TopHat's count of “spanning mate pairs where one end spans a fusion,” which is sometimes referred to as ‘spanning and supporting reads’).

The breakpoint sequence reported by the callers was trimmed to include 50 bases on each side of the fusion and consolidated into one column—Breakpoint Sequence. The fusion breakpoint is delineated by a “|”. Note that this is the breakpoint sequence as inferred by the caller, and is not simply obtained from the reference genome. Because the inferred sequence may reflect actual sequence observed by the spanning reads, this sequence may represent the complement of the reference genome sequence.

Since neither of the callers provides a definitive ‘5-prime’ or ‘3-prime’ flag, we infer the relative 5′-3′ orientation of the fusion partners by combining a caller parameter with the gene strand annotation. For deFuse, the orientation was inferred for each partner based on the following combination of the gene strand and the deFuse output property ‘genomic_strand:’

TABLE 28 Gene deFuse_genomic_strand Strand + − + 5′ 3′ − 3′ 5′

TopHat reports a different metric—the relative orientation of reads mapped to the gene partners, so a different rule set is required for inferring 5′-3′ order for a pair of genes:

TABLE 29 tophat_orientation Gene A/B Strand ff fr rr Rf +/+ 5′-3′ 3′-5′ +/− 5′-3′ 3′-5′ −/− 3′-5′ 5′-3′ −/+ 3′-5′ 5′-3′

A Valid Orientation field was labeled as “Y” if there was an inferred 5′ and 3′ partner for a given gene fusion call.

RepeatMasker Annotation.

Each predicted breakpoint location was also annotated with RepeatMasker features in the neighborhood of the breakpoint. This was done to identify breakpoints in highly repetitive genomic regions, where alignment errors were likely to affect the prediction of the chimeric transcript.

Specifically, a 25 bp sequence upstream or downstream of the 5′ and the 3′ partner breakpoint respectively was selected as a ‘breakpoint flank’. These flanks were intersected against the RepeatMasker elements set (www.repeatmasker.org) downloaded from UCSC Table Browser on Aug. 24, 2012. We reported the element name, element length, and amount of overlap with the 26 base breakpoint flank region for each breakpoint. Currently, the RepeatMasker elements are not filtered for specific element types (LINES, SINES, simple repeats, etc.).

For each fusion prediction, we set a RepeatMasker Overlap field to equal the number of bases the breakpoint flank sequences overlaps with a RepeatMasker element, and considered overlaps of 12 or more bases to be significant. The frequency of significantly overlapping fusion calls is used in the Oncomine Prioritization described below such that gene fusions with a lower frequency of overlap are considered higher quality.

Fusion Exon Expression Imbalance.

Fusions were visualized using RNASeq exon expression data to provide secondary evidence of true positive fusion events by searching for exon expression imbalance before and after the breakpoint call. Specifically, if the 3′ partner's expression is impacted by the 5′ partner's promoter region, then exon expression should increase post-predicted breakpoint. This effect is especially visible when viewing fused versus non-fused patient samples.

TCGA Exon Expression Data.

TOGA exon expression data was downloaded from the Broad's GDAC Firehose site. The RPKM RNASeq values are listed for each patient as Gene Annotation Format (GAF) features corresponding to a composite of UCSC exons from several different gene definitions including RefSeq. After downloading data for 21 diseases, we found that 4 different sets of GAF features were used to annotate RPKM expression. Finally, availability of patient expression data varied per disease in V1 and V2 RNASeq analysis pipelines described above.

To address these challenges we first mapped UCSC RefSeq exons to available GAF features and calculated the percentage overlap between each RefSeq exon and GAF feature. This step is critical since all CBI processed fusion breakpoints are mapped to UCSC Refgene definitions downloaded on Jul. 18, 2012 and these breakpoints must in turn be mapped to GAF features. 80.8% of the 396,298 RefSeq exons map perfectly to GAF features in the plot shown below. We selected and reported on the RefSeq exon and GAF feature pair that resulted in the largest overlap.

A value called rg_pct provides a metric of the mapping quality of a given RefSeq exon with a GAF feature based on the following formula: rg_pct=overlap/length_(refseq)*overlap/length_(GAF feature)

Mappings with an rg_pct value of 1 overlap perfectly, while values less than 1 indicate the RefSeq exon or GAF feature did not map to the exact same genomic regions and the RPKM value may be suspect.

We selected RNASeq V2 data for all diseases except STAD due to non-availability of V2 data.

Cell Line Exon Expression Data.

Exon expression data for cell line samples was generated from the COLE BAM files obtained from CGHub. The method employed was similar to Step 18 as described in the “TOGA mRNA-seq Pipeline for UNC data” method available here: webshare.bioinf.unc.edu/public/mRNAseq_TCGA/UNC_m RNAseq_summary.pdf.

A difference between the UNC method and our method is the use of RefSeq Exons BED in our method instead of a composite exons BED used by the TOGA.

Exon Expression Imbalance Calculation.

Each sample was systematically analyzed for evidence of potential 5′ promoter-induced imbalance in 3′ partner expression. Expression levels for each gene were first converted to a log scale, and then z-score normalized across each disease's sample cohort. This normalization was performed at the exon level to account for population-wide trends such as 3′ bias or poor RefSeq exon/GAF feature match (see below).

Raw RPKM expression values (top) vs. z-score normalized values for PLXNB21 and COL7A1 in Ovarian Serous Carcinoma patients (See FIG. 8 A-D). The population-wide dips in PLXNB1 expression at exons 12, 17 and 23 are smoothed out in the normalized data. A sample predicted to harbor a fusion between these genes is highlighted in red; wild-type patients are shown in blue. The red diamond indicates the caller-predicted breakpoint exon.

Prior to normalization, samples that were considered wild-type for the fusion under consideration but that were predicted to harbor other fusions involving one of the gene partners were removed from the wild-type population, so as not to contaminate z-score calculations.

After normalization, each sample was assigned a p-value calculated via one-sided Student's t-test on the hypothesis that the sample's post-breakpoint normalized expression values (Population A) have a higher mean than the pre-breakpoint values (H₀: μ_(A)≤μ_(B)). The caller-predicted breakpoint was used to separate the expression populations for samples identified by either fusion caller.

P-values were also calculated for each wild-type sample to facilitate analysis of p-values for fusion-positive samples in the context of the overall population. This allows us to discard fusions involving genes that exhibit population-wide exon imbalance trends that are not fusion-induced. Any sample whose p-value did not rank within the top fraction of wild-type sample p-values was discarded. The breakpoint that maximized the difference between pre- and post-breakpoint expression levels was used for wild-type sample p-value calculation.

Fusion Summarization.

Fusions were summarized within a disease based on the occurrence of unique gene pairs, and based on the occurrence of individual genes, possibly with multiple partners.

For a unique fusion pair (unique by Entrez ID pair), the number of samples within a disease with at least one prediction of that fusion by either caller is the Fused Sample Count. Since multiple breakpoints for the same pair of genes may be reported in one sample and across the samples, the number of unique fusion pairs within each disease is much less than the total number of fusion calls. In order to filter and prioritize fusions at the gene pair level rather than the fusion call level, several of the fusion caller properties were summarized. The following table shows the properties that were summarized for a given fusion partner pair across the individual predictions:

TABLE 30 Property Summary Method DEFUSE_EVERSION % of total fusion calls = ‘Y’ DEFUSE_VALID_ORIENTATION % of total fusion calls = ‘Y’ DEFUSE_NUM_MULTI_MAP % of total fusion calls >0 TOPHAT_VALID_ORIENTATION % of total fusion calls = ‘Y’ 3P/5P_REPEATMASKER_OVERLAP % of total fusion calls ≥12

The Adjacent flag is set for a fusion if the genes are <1 Mb apart on the genome and the defuse_eversion flag is set in 75% of the individual fusion prediction for these fusion partners.

Gene-Level Summary.

Fused sample counts were also summarized at the gene level (unique by Entrez gene ID) within each disease type and across diseases (pan-cancer). This summarization approach was irrespective of inferred orientation within the fusion. In addition, fused sample counts were tallied for only the Oncomine Priority fusions (described below).

Individual unique fusion pairs were cross-referenced to the Mitelman database of genomic aberrations (cqap.nci.nih.gov/Chromosomes/Mitelman). The match was done based on gene names and not disease type. Therefore, gene fusions reported in Mitelman in a certain disease may have occurred in a different disease type in the TOGA datasets.

Gene fusions summarized at the gene level were cross-referenced to the Mitelman database based on gene name. Thus, there is more potential for the gene as reported in Mitelman to be of different histology or altogether different aberration type (for example a large chromosome-level deletion instead of a fusion) than the predicted unique fusion pairs.

Normal Sample Fusion Blacklist.

With the assumption that all fusions called in TOGA normal samples are false positives, we asked the following questions: 1) Are fusion calls in tumor samples identified in normal samples? 2) Are Oncomine Prioritized Fusions identified in tumor samples also identified in normal samples? Answering the first question provides a baseline sense of the technical false positive rate in tumor gene fusion calls. The second question is a sanity check on how well the Oncomine Priority Fusion filter is overcoming this problem. 344 paired-end normal samples across 10 diseases were downloaded and processed using the same deFuse pipeline described above. A total of 56,579 total fusion calls consisting of 6,024 unique fusions were observed. These normal sample fusion calls were used to generate a blacklist and remove these false positives from Oncomine Priority gene fusions.

Paralogous Fusion Partner Blacklist.

A blacklist of fusions between paralogous gene family members was assembled using two strategies: 1) manually inspecting high frequency fusion partner gene names and 2) comparing the first 3 characters of all Priority Fusion partner gene names. In the latter strategy, fusion partners were verified to be “paralogous” using HomoloGene, Ensembl, SIMAP, and GeneDecks V3 before inclusion in the final blacklist. The table below shows the top 10 most commonly observed gene fusion calls between paralogous fusion partners. The entire table consists of more than 400 unique paralogous gene fusions and is used to remove these false positives from our Oncomine Priority gene fusions.

TABLE 31 GeneA GeneB Observed in Symbol Symbol Normal TCGA Cancer Types HLA-B HLA-C YES BLCA, BRCA, CESC, COAD, GBM, HNSC, KICH, KIRC, KIRP, LAML, LGG, LIHC, LUAD, LUSC, OV, PAAD, PRAD, SKCM, STAD, THCA, UCEC HLA-A HLA-B YES BLCA, BRCA, CESC, GBM, HNSC, KICH, KIRC, KIRP, LAML, LGG, LIHC, LUAD, LUSC, OV, PAAD, PRAD, SKCM, STAD, THCA HLA-A HLA-C YES BLCA, BRCA, CESC, GBM, HNSC, KICH, KIRC, KIRP, LAML, LGG, LIHC, LUAD, LUSC, OV, PAAD, PRAD, SKCM, STAD, THCA TTLL12 TTLL12 YES BLCA, BRCA, CESC, GBM, HNSC, KICH, KIRC, KIRP, LAML, LGG, LIHC, LUAD, LUSC, OV, PAAD, PRAD, SKCM, STAD, THCA TRPV1 TRPV1 YES BLCA, BRCA, CESC, GBM, HNSC, KICH, KIRC, KIRP, LAML, LGG, LIHC, LUAD, LUSC, OV, PAAD, PRAD, SKCM, STAD, THCA B9D1 B9D1 YES BLCA, BRCA, CESC, GBM, HNSC, KICH, KIRC, KIRP, LGG, LIHC, LUAD, LUSC, OV, PAAD, PRAD, SKCM, THCA TGIF2- TGIF2- YES BLCA, BRCA, CESC, GBM, HNSC, KICH, KIRC, KIRP, C20ORF24 C20ORF24 LAML, LGG, LIHC, LUAD, LUSC, OV, PRAD, SKCM, STAD, THCA HLA-B HLA-E YES BLCA, BRCA, CESC, COAD, GBM, HNSC, KICH, KIRC, KIRP, LAML, LGG, LIHC, LUAD, LUSC, OV, PAAD, PRAD, READ, SKCM, STAD, THCA, UCEC SEC16A SEC16A YES BRCA, CESC, GBM, HNSC, KICH, KIRC, KIRP, LAML, LGG, LUAD, LUSC, OV, PRAD, SKCM, THCA LOC390940 LOC390940 YES BLCA, BRCA, CESC, GBM, HNSC, KICH, KIRC, KIRP, LGG, LUAD, LUSC, OV, SKCM, STAD, THCA

Fusion Prioritization—Oncomine Priority Scheme.

The Oncomine Priority scheme outlined below was designed by iterative exploration of the top results in the Level I fusion predictions and systematic elimination of suspect false-positive fusions, while retaining previously discovered ‘true-positive’ (Mitelman) fusions. This scheme was meant to highlight fusions that conformed to certain features expected of a ‘true-positive’ fusion, and conversely, lack features observed in many ‘false-positive’ fusions.

A fusion is an Oncomine Priority fusion if:

TABLE 32 Fusion Summary Property Value Explanation DEFUSE_VALID_ORIENTATIONTOPHAT_VALID_ORIENTATION >0.75 Most predictions in correct orientation ADJACENT ‘N’ REPEATMASKER_FREQUENCY <0.25 Minority or none of predicted breakpoints are in repetitive regions DEFUSE_NUM_MULTI_MAP >0 Most spanning reads map uniquely to fusion breakpoint PARALOGOUS_PARTNERS Not on Manually curated blacklist of predicted fusions Paralogous between paralogous genes Blacklist OBSERVED_IN_NORMAL Not on Normal List derived from processing 344 Normal samples Blacklist using deFuse.

Example 6: Oncomine NGS Mutation Methods

Mutation Integration.

The goal of the data integration was to create the most complete set of NGS mutation data currently available. We considered the following sources:

Primary Data Sources

COSMIC Cell Lines Project

TCGA Data from Broad GDAC Mutation_Packager (stddata build)

TCGA Data from DCC level 2

Compendia mutation calls based on TCGA Data

Publications containing NGS mutation data

COSMIC Cell Lines Project

The Cancer Genome Project has characterized the exomes of over 1000 cancer cell lines for mutations. The database provides the mutation data, filtered for quality, in a flat-file format. The cell line data was subjected to the same Oncomine curation and annotation processes used for clinical mutation data. Cell line names were vetted against the Oncomine ontology, and cancer types were standardized to be comparable with clinical mutation data.

The dataset was obtained from the Wellcome Trust Sanger Institute Cell Lines Project website: cancersangerac.uk/cancergenome/projects/cell_lines as it appeared in November 2013.

Broad GDAC Mutation_Packager. Broad has been working since Q3 2011 on gathering and integrating mutation data from multiple sources.

docs.google.com/document/d/18XIWv-a9xLBOflNikOa9rCXOyiravMM8-PVJxAQPPo/edit

The above document details the provenance of the MAF files the Broad integrates into Mutation_Packager standard data runs. The Broad has integrated many MAF files that are maintained outside of the central TCGA DCC system, often by members of the Analysis Working Groups themselves. We have performed extensive comparisons between all MAF files available to us. It is our belief that the Broad has the most complete mutation data available.

For this release, we integrated data from the 2013_08_09 stddata build.

TCGA DCC Level 2. This is the controlled access mutation data available from the DCC. TCGA has a page on their wiki that provides additional details about the MAF files available:

wiki.nci.nih.gov/display/TCGA/TCGA+MAF+Files

For this release, we considered all MAF files available as of Sep. 15, 2013.

NGS DNASeq Mutation Calls. PRAD mutation calls available from TCGA were of low quality and resulted in false-positive ‘Gain of Function’ predictions. Therefore, all calls for this disease were sourced from Compendia's own mutation calling pipeline. The Compendia mutation calls were made to conform to the MAF file format for integration. Please see the Appendix: Compendia NGS DNASeq Mutation Calling for more details. Included in this release are 170 Prostate Adenocarcinoma patients.

Hand-Curation of All NGS Data. TCGA and Non-TCGA NGS datasets were sourced by the Oncomine curation team directly from their primary sources—mainly peer-reviewed cancer publications and the above publically accessible databases. Mutation data, usually available in the Supplementary Materials, was brought to the standard required for mutation re-annotation and classification as part of the overall NGS Mutation processing pipeline. Cancer types were curated using the Oncomine cancer type ontology, assigning the appropriate Oncomine Cancer Type based on the best-available clinical metadata present in the publication. Since all the published experiments claimed whole-genome (‘NGS’) coverage, the null gene set for each dataset was assumed to be inclusive of all human RefSeq genes. The non-TCGA data was processed in the same exact way as the TCGA MAF-file data for the rest of the mutation analysis pipeline.

Remove Duplicate Mutations. We performed some simple clean-up operations to remove duplicate mutation records present in the source data. We also performed several file-column name re-mappings, as many of the sources do not adhere to the MAF file standard. Duplicate mutations from various tumor/normal aliquot pairs of the same patient sample were removed.

Mutation Annotation. Data obtained from the TCGA and non-TCGA sources contains mutation results from datasets processed and annotated by different genome sequencing centers or authors over the course of several years. This leads to the mutation calls annotated using different gene models and using different conventions for variant classification. Since Compendia's approach to defining mutations relies on accurate variant annotation, we re-annotated the mutations against a single set of transcripts and consistent variant classification rules. A standard annotation pipeline ensured that mutations across disease types are evaluated consistently and are subject to common interpretation during the nomination of potential oncogenes or tumor suppressor genes. It also provided important annotation not consistently available from the primary sources, such as the HGVS-style mutation nomenclature (e.g., V600E).

Mutations obtained from primary sources are processed by Compendia according to the following general steps (details provided below).

We first re-annotated each mutation using Compendia's Oncomine transcript set. Successfully annotated mutations received Compendia-derived annotation, while the rest retain annotation obtained from the primary source. Annotation includes:

Variant classification

Variant position

Variant change.

Several filtering steps are implemented to remove redundant annotation in multiple transcripts, and mutations located outside of gene regions of interest.

Excluding “Ultra-mutator” Samples. In certain diseases, such as Endometrial Carcinoma, several highly-mutated samples may dominate the overall mutation counts. We also observed such “ultra-mutator” samples in Lung Adenocarcinoma, Gastric cancer, Melanoma, and Colorectal cancer. Based on a cut-off determined by analyzing ulta-mutator outliers in several cancer types, we decided on <5,000 non-silent exon mutations as the threshold for inclusion of a sample in our recurrence analysis. We therefore excluded a number of ultra-mutator samples in this dataset from our downstream analysis pipelines.

In the Mutation Annotation step, we attempted to re-annotate the mutations obtained from the primary sources against a standard transcript set compiled by Compendia. This transcript set included RefGene transcripts from hg18 and hg19 genome builds, obtained from UCSC on Feb. 19, 2012.

Each mutation is individually mapped against a contig in the Oncomine Transcript Set within the specified genome build. SNP mutations were mapped directly to their start location, while for small insertion (INS) and deletion (DEL) mutations a position of interest is selected for mapping. For insertions, the position of interest is the base at which the insertion occurred. Depending on the direction of the transcript, this can either be the start or the end coordinate of the mutation, depending on whether the gene is on the positive or negative strand respectively. For deletions, the position of interest is the deleted base if the transcript is on the positive strand or the last base deleted if the transcript is on the negative strand. This adjustment ensures that the mutation position is defined as the first base affected by the insertion/deletion with respect to the direction of the transcript translation, i.e. 5′→3′.

For a mutation successfully mapped to a transcript, the Compendia mutation annotation was inferred with respect to that transcript. For mutations that failed to map, the annotation from the primary data source was retained, and a variant position for Hotspot calculations was constructed based on the genomic coordinate (more details below). Since only the standard set of 23 chromosomes was included in our transcript set, mutations located on mitochondrial or other non-standard contigs were not mapped.

Below is a description of the criteria used in annotating the mutations that map to the Oncomine Transcript Set.

Variant Classification. For each mutation successfully mapped to a transcript, the variant classification was inferred using a combination of mutation and annotation properties. Our approach identified six main mutation variant classifications, all located within transcript. Variant classifications for mutations outside a gene region (e.g. intergenic) are currently not considered (see filtering section below). The following are the criteria used for inferring the variant classification:

TABLE 33 Transcript Variant Classification Criteria Region Splice_Site Mutation is within 2 bp of a splice site exon or intron 3′UTR, 5′UTR Mutation is in UTR region and not within UTR exon 2 bp of splice site Intron Mutation is in an intron and is between intron 3 to 10 bp from a splice site Missense, Nonsense, Nonstop, Mutation is a SNP coding exon Silent Frame_Shift_Ins/Del Mutation is an INS/DEL not divisible by 3 coding exon In_Frame_Ins/Del Mutation is an INS/DEL divisible by 3 coding exon Non_Coding_Exon Mutation is in a non-coding transcript non-coding exon

This list of variant classifications is a subset of the allowed variant classification specified by the TOGA for the MAF file format.

wiki.nci.nih.gov/display/TCGA/Mutation+Annotation+Format+%28MAF %29+Specification

This subset covers the mutation classes of interest for recurrence analysis and identification of potential Gain or Loss of Function genes, and is thus sufficient for the vast majority of the mutations that are mapped to the Oncomine Transcript Set. The following table describes the likely variant classification that would be assigned versus an original author classification (assuming mutation maps to the same transcript as that used in defining classification), and the relative abundance of that type of mutation in the source dataset:

TABLE 34 Potential Oncomine Mutation Classification (H)otspot, Example TCGA Variant Equivalent Compendia (D)eleterious Classification Variant Classification or (O)ther Missense_Mutation Missense_Mutation H, O Nonsense_Mutation Nonsense_Mutation D Nonstop_Mutation Nonstop_Mutation H, O Silent Silent O Frame_Shift_Del Frame_Shift_Del D Frame_Shift_Ins Frame_Shift_Ins D Translation_Start_Site Missense_Mutation O In_Frame_Del In_Frame_Del H, O In_Frame_Ins In_Frame_Ins H, O 3′UTR 3′UTR O 5′UTR 5′UTR O Non_coding_exon (or Non_coding_exon H, O “RNA”) Splice_Site Splice_Site O Intron Intron — 5′Flank --not supported — by Oncomine transcript set-- IGR --not supported — by Oncomine transcript set-- Other (classification present — in mutation list but not supported by TCGA)

Variant Position.

One of the primary goals of the current analysis is to identify genes with Hotspot mutations, which are mutations of a certain classification that are observed at the same location in multiple tumor samples. To effectively identify recurrence and define a hotspot for each mutation, we must construct a mutation spot identifier that encompasses the mutation position, the identity of the amino acid or base affected, and the variant classification. We aggregated mutations that occur at the same location irrespective of the specific base change they generate. Therefore, we only used the reference base or amino acid to define the variant position. This ensures that mutations affecting the same codon or genomic position will be counted towards a possible hotspot, even if the alternate alleles they generate are different. For example, for a given gene, missense mutations V600E, V600F and V600G would all have a variant position of V600 and would thus be aggregated together when identifying hotspot mutations. Our variant position is thus defined as follows: Variant Position=mutation spot{baselcodon}+reference{base|AA}+[variant classification]

If the mutation is in a coding region, then the codon number and the respective amino acid at the base of interest is used to identify the mutation spot—p.L116_in_frame_del—for example. If the mutation is in a non-coding region, such as the UTR, then the position and identity of the reference nucleotide at the base of interest is used to identify the mutation spot—c.110C—for example.

For Splice_Site mutations outside of the coding region, the variant position is specified relative to the splice boundary. The relative position is identified using a +{1 |2} or a −{1 |2} (splice site mutations are those within 2 bases of a splice junction). As with insertions and deletions, a suffix of “_Splice_Site” is added for a Splice_Site mutation. For INS and DEL mutations, a suffix indicating an in frame (“_in_frame_ins” or “_in_frame_del”) or frame shift (“_frame_shift_ins” or “_frame_shift_del”) is added to the variant position.

In summary, the following are examples of the different possible variant position formats:

TABLE 35 Variant Near Splice In Coding Type Site? Region? Variant Position SNP YES YES p.A42_Splice_Site NO c.42 + 1_Splice_Site SNP NO YES p.A42 (Missense, Nonstop, Silent) p.Stop42 (Nonsense) NO c.*42T (3′UTR) c.-42C (5′UTR) c.42 (Non_coding_exon) INS YES YES p.A42_Splice_Site NO c.42 + 1_Splice_Site NO YES p.A42_{in_frame_ins|frame_shift_ins} NO c.*42G_{in_frame_ins|frame_shift_ins} (3′UTR) c.-42G_{in_frame_ins|frame_shift_ins} (5′UTR) c.42 (Non_coding_exon) DEL YES YES p.A42_Splice_Site NO c.42 + 1_Splice_Site NO YES p.A42_{in_frame_del|frame_shift_del} NO c.*42T_{in_frame_del|frame_shift_del} (3′UTR) c.-42C_{in_frame_del|frame_shift_del} (5′UTR) c.42 (Non_coding_exon)

For mutations that do not map to the Oncomine Transcript Set, and hence do not have a transcript-based location, the genomic location (start position) and the reference nucleotide (reference allele) is used as the variant position irrespective of the coding region or splice site proximity. The variant classification supplied by the primary data is then added as a suffix. For example, a SNP missense mutation would have a variant position such as “chr19_c.C22952756_Missesnse_Mutation”, and a splice site SNP would have a variant position “chrl_c.A155025094_Splice_Site”. The variant change (see below) for these mutations is not defined.

Although the suffix of the variant position often implicitly incorporated the variant classification, when calculating hotspots, both the variant position and the variant classification are explicitly used for aggregating mutations. Therefore, mutations that may produce identical variant positions but have different variant classifications (such as a missense and a nonsense SNP) were tallied separately.

Variant Change.

The variant change provides HGVS-like information about the alternate allele change of the mutation. For SNP mutations in the coding region, the variant change is a full HGVS protein-level sequence variant description, indicating the alternate amino acid. For SNPs outside of the coding region, the alternate allele nucleotide base is provided.

For INS and DEL variant types, the variant position (see above) was used as the variant change. In these cases, the consequence of the change at the amino acid level is not inferred. As such, variant change for INS/DEL does not strictly follow HGVS specification.

The following are illustrative examples of variant changes for Compendia-derived mutation annotation:

TABLE 36 Variant Mutation Classification Variant Position Variant Change SNP in CDS, Missense_Mutation p.E137 p.E137K E > K, residue 137 SNP in Intron Splice_Site c.4913 − 1_splice_site c.4913 − 1 C > 2, two bp from splice site INS in CDS at Frame_Shift_ins p.G264_frame_shift_ins p.G264_frame_shift_ins residue Gly 264 DEL of one 3′UTR c.*1007A_frame_shift_del c.*1007A_frame_shift_del base in a UTR

For mutations that do not map to the Oncomine Transcript Set, the variant classification from the primary data source was retained.

Transcript Filter.

To avoid retrieving multiple transcripts, and hence multiple annotations for a single mutation within a gene, we kept only one transcript per mutation per gene (unique Entrez ID). If a mutation mapped to several transcripts of a gene, only one was chosen. However, if a mutation mapped to several genes, then only one transcript per gene was selected. It is thus possible for a mutation to receive two different annotations, but only if they stemmed from transcripts with different Entrez IDs.

We chose the representative transcript for a mutation based on the following priority scheme:

Transcript with the most impactful variant classification:

High impact in coding: Missense, Nonsense, Nonstop, Frame-shift

Low impact in coding: In-frame, silent

Outside of coding region: Splice Site, 3′ or 5′ UTR, Non-coding exon

Outside of exon: Intron

If there is a tie based on priority, the transcript with the shortest (by length) RefSeq transcript accession is chosen, followed by the alphanumerically smallest transcript accession in event of further ties. For example—of the transcripts NM_003319, NM_133378, and NM_00125685 for the TTN gene, we would choose NM_003319 as the representative transcript.

These steps allowed us to repeatedly choose a consistent transcript for the same type of mutation at one location. One consequence of choosing the most impactful transcript is that multiple transcripts may be utilized for mutations at multiple locations in a single gene. However, the benefit of this scheme is that any mutations of the same variant classification at the same location are always assigned to the same transcript, and hence will be in the same frame of reference when computing recurrence for hotspot identification.

Filter by Mutation Class and Type.

All mutations were further filtered by variant type and class. To avoid including mutations of minor interest to gene function analysis, we filtered out mutations that were not resolved to a gene region, either because they fell significantly far outside of a transcript, or because they were in a location not associated with a RefSeq gene. These mutations were evident either by their lack of gene identifier (Entrez ID=0 or blank), or membership in the following variant classes: Intron, 5′Flank, IGR, and miRNA.

We also filtered out mutations with variant type DNP, TNP, ONP, Complex_substitution, and Indel, as their annotation was not supported by our pipeline

Since certain data sources included extensive amounts of intronic and intergenic mutations, this filtering step significantly reduces the size of the dataset as many NGS datasets don't apply these filters pre-publication.

Classifying Mutations as Hotspot, Deleterious, or Other.

The next step in our analysis pipeline identified recurring mutations in multiple samples based on their variant position, and categorized them into Hotspot, Deleterious or Other variant categories. For this step, and the subsequent frequency calculations, mutations for each disease type were processed independently. Only mutations of the same variant classification were tallied together, so, for example, a missense mutation and a silent mutation at the same position are counted separately.

To identify driver events, each mutation for a given Entrez Gene ID was categorized as “Deleterious” or “Hotspot” depending on the following criteria:

A mutation was deemed ‘recurrent’ if it was observed in the same variant position in 3 or more tumor samples.

A mutation belongs to the “Hotspot” variant category if it is:

Recurrent AND

Annotated with one of the following variant classifications:

-   -   In-frame insertion/deletion     -   Nonstop     -   Missense     -   Non_Coding_Exon

A mutation belongs to the “Deleterious” category if it is:

Non-recurrent AND

Annotated with one of the following variant classifications:

-   -   Frame shift insertion/deletion     -   Nonsense

A mutation is considered in the “Other” variant category if it did not fit the above criteria.

The Oncomine Mutation Classification and the Variant Classification can be used to summarize the relative frequencies of various mutations at the gene level.

Nominating “Gain of Function” and “Loss of Function” Genes.

Individual genes were classified into predicted functional classes, namely “Gain of Function”, “Recurrent Other”, and “Loss of Function”, to reflect their relative enrichment in potential activating or deleterious mutations. Details of the scheme used to make the classification are provided below.

Mutated Sample Frequency Calculation.

Mutation frequencies for each gene were calculated with respect to a given variant classification and variant category across all samples within a disease type. Overall mutation frequency for a gene within a disease was calculated by combining mutations of all variant classifications.

Overall Mutation Frequency.

Overall mutation frequency for a gene was obtained by dividing the total number of samples with at least one mutation of any variant classification in that gene (Mutated Sample Count) by the total number of samples in the given cancer type (Sample Count).

Hotspot Frequency.

Hotspot frequency for a gene was obtained by dividing the total number of samples with at least one mutation belonging to the “Hotspot” Oncomine Mutation Classification by the Mutated Sample Count—the total number of samples with at least one mutation for the given gene. If a sample had both Hotspot Missense and a Hotspot In-Frame Deletion, for example, it would only be counted once.

Hotspot Missense Frequency.

To obtain a Hotspot Missense Frequency for a gene, the number of samples containing at least one Missense mutation with an Oncomine Mutation Classification of “Hotspot” was divided by the Mutated Sample Count—the number samples with at least one mutation of any type in this gene. Samples with more than one mutation of such type were only counted once.

Deleterious Frequency.

To obtain the Deleterious frequency for a gene, the number of samples containing at least one mutation with an Oncomine Mutation Classification of “Deleterious” was divided by the Mutated Sample Count—the number of samples with at least one mutation for the given gene. Samples with more than one mutation of that type were only counted once.

Other Frequency.

To obtain the Other frequency for a gene, the total number of samples with at least one mutation with an Oncomine Mutation Classification “Other” was divided by the Mutated Sample Count—the total number of samples with at least one mutation for the given gene. If a sample contained both splice site and UTR mutations, for example, it would only be counted once.

Hotspot, Other, and Deleterious Frequency Consideration.

Hotspot, Other, and Deleterious frequencies should not be expected to add up to 100%, since a sample may have been counted in more than one of these categories.

Assessing Significance of Hotspot and Deleterious Mutations.

The Hotspot and Deleterious p-values for each gene within a disease are calculated by two independent methods.

Significance of Deleterious Mutation Enrichment.

To assess whether a gene was significantly enriched for deleterious mutations compared with other genes, given the background mutation rate, we performed Fisher's exact test using the following contingency table:

TABLE 37 Deleterious Other Gene of Interest A B All Other Genes C D

where A, B, C, and D are counts of mutations across a disease. Nonsense mutations, frame shift insertions and frame shift deletions are classified as deleterious mutations, while mutations of any other type (UTR, silent, missense, etc., but non-intergenic) count as others.

Q-values are calculated within each disease, by counting the number of genes with deleterious mutations (N), and calculating the rank of each association. The q-value for a given p-value is then Q=p*N/rank.

Significance of Recurrent Hotspot Mutations.

In order to calculate gene-specific p-values, the significance of the most recurrent hotspot on that gene is assessed. Given the assumption that each sequence position was equally likely to mutate, each gene can be tested whether the most recurrent is significantly greater than that expected using a multinomial test. This is an exact test of the sampling algorithm that has been implemented in previous versions. One of the advantages of this test is that the p-value precision is increased to 1E-16, so no flooring occurs. To obtain hotspot mutations, we filtered the mutations to remove any that did not affect the coding sequence (i.e. by removing silent, UTR, stop codon, and splice site mutations), and then removed mutation data for genes that we could not annotate with RefSeq transcript identifier. We then counted the mutations observed for each transcript in each disease. We calculated the amino acid sequence length by dividing the CDS length by three and subtracting 1.

The exact calculation of the p-value is framed as the following. Given an amino acid sequence of length x, an observed number of hotspot mutations n, what is the likelihood of observing r or more mutations at the most recurrent spot by chance For each gene, the p-value is calculated by the following formula:

$\begin{matrix} {p = {\Pr\left( {y_{(X)} \geq r} \right)}} \\ {= {1 - {\Pr\left( {{y_{1} < r},{y_{2} < r},\ldots,{y_{X} < r}} \right)}}} \\ {= {1 - {\sum\limits_{0}^{r - 1}{\frac{n!}{{y_{1}!}{y_{2}!}\ldots{y_{X}!}}\left( {1/x} \right)^{n}}}}} \end{matrix}$

where y_((X)) is the mutation count at the most recurrent hotspot, and y₁, . . . , y_(X) stands for the mutation count at each spot 1, . . . , x.

When n and x are large, the above formula can be very slow, an approximation with Bonferroni-Mallows (BM) bounds were used:

$1 - {\frac{n!}{n^{n}e^{- n}}\left\{ {\prod\limits_{i = 1}^{X}{P\left( {y_{i} \leq {r - 1}} \right)}} \right\}{P\left( {W = n} \right)}}$

where y_(i) is a Poisson random variable with mean n/x, and W=Σ_(i=1) ^(X), where Y is a truncated Poisson. P(W=n) is estimated by Edgeworth Expansion. The lower and upper Bonferroni-Mallows bounds for the p-value are:

${1 - {{Binomial}\left( {{r - 1},n,\frac{1}{x}} \right)}^{x}} \leq p \leq {x*{\left( {1 - {{Binomial}\left( {{r - 1},n,\frac{1}{x}} \right)}} \right).}}$

If the approximation falls outside of the BM bounds, either the lower bound or upper bound was used. It rarely occurred in our data, and it mostly occurred for small p-values (p<1e-16) or large p-values (p˜=1).

Q-values are calculated using the Benjamini-Hochberg method, which is Q=p*N/rank, where N is the number of transcripts and rank is the rank of each p-value.

Silent Hotspot Mutations.

Recurrent silent mutations—silent hotspots—seem to be an indication of sequencing errors, occurring in regions of low sequence quality and serving as a ‘canary in the coal mine’ for false-positive missense mutation peaks in the neighboring nucleotides. Based on reviewing genes with silent hotspots, and the evaluation of neighboring silent peaks, we believe that these genes are subject to systematic sequencing errors, and hotspot mutations in these genes should not contribute to the gene classification.

Oncomine Gene Classification Rules.

Once the mutations have been classified, individual genes are nominated to one of three classes—“Gain of Function,” “Loss of Function,” and “Recurrent Other.” The classification is based on the combination of relative frequencies and the significance of the mutations observed in the gene. The significance of the mutations per gene is assessed by a p-value.

Recurrent Silent Mutations.

A “Gain of Function” gene will have a relatively high frequency of Hotspot Missense mutations and a low frequency of Deleterious mutations, while a “Loss of Function” gene contains a large fraction of Deleterious mutations. “Recurrent Other” genes tend to contain recurrent insertion/deletion mutations, some of which—for example recurrent frame shift indels of 1 base—exhibit signs of potential false-positive calls that may arise from local alignment errors. In general, we are more confident about the functional importance of genes classified as Gain/Loss of Function.

Pan-Cancer Analysis.

To summarize mutations across diseases we performed identical calculations as we did for within-disease analyses, but without stratifying the mutation records by disease. All mutation records were aggregated, and frequencies, variant categories and gene classes were calculated in this pan-cancer context. For the pan-cancer summary, genes (unique by Entrez ID) are summarized across all diseases with one row per disease. However, a summary of the genes within disease is also provided, but in a pan-cancer context. This means, for example, that samples with Hotspot mutations are totaled within a disease, but only for the mutations considered Hotspots in a pan-cancer context. Cancer types with <20 samples were included in Pan-Cancer analysis, even though they were not eligible for within-disease analysis due to low sample count.

Cell Line Annotations.

Cell line mutation data was subjected to the same Oncomine curation and annotation processes described above except for mutation and gene classification. Instead, mutations from cell lines were annotated with Oncomine mutation classification and gene classifications whenever a mutation in a cell line was also observed in a clinical sample. This annotation was performed only for mutations having a Hotspot or Deleterious or Other Oncomine mutation classification. If a mutation was not observed in tumors, it would receive “Unobserved in Tumor” mutation classification.

Mutations from a cell line and a tumor sample are considered equivalent if they belong to the same gene, and have the same variant position and variant classification.

Cell lines names were vetted against internal Oncomine ontology, and cell line cancer types were standardized to be comparable with clinical mutation data. Several cell lines whose identity or cancer type could not be independently verified through databases or publications were removed from our analysis. The mutation annotation from clinical data was performed in a pan-cancer and within-disease contexts.

NGS DNASeq Mutation Calling

BAM File Selection.

We queried TCGA's CGHub to identify patients having a single tumor-normal BAM pair. We did so to remove the possibility of mutation call differences due to different tumor-normal pairs.

Reference Genome Builds.

We identified the reference genome builds used to align the reads in the BAM files by parsing the SAM headers. We located, downloaded, and indexed all the reference genome builds which are needed as inputs to the mutation caller packages.

Mutation Calling.

We employed the following somatic mutation calling packages for this analysis:

MuTect (1.0.27783), Broad Institute, Cancer Genome Analysis Group (CGA) (Cibulskis, 2013)

SomaticIndelDetector (1.6-13-g91f02df), Broad Institute, Genome Analysis Toolkit (GATK)

MuTect.

MuTect performs initial preprocessing to remove “reads with too many mismatches or very low quality scores” (MuTect documentation). Next, for a candidate mutation two log odds (LOD) scores are calculated that describe the likelihood of a mutation being present in the tumor sample (LOD_(T)) and not mutated in the normal sample (LOD_(N)):

${{LOD}_{T} = {\log_{10}\left( \frac{\begin{matrix} {P\left( \left. {{observed}{data}{in}{tumor}} \right| \right.} \\ \left. {{site}{is}{mutated}} \right) \end{matrix}}{\begin{matrix} {P\left( \left. {{observed}{data}{in}{tumor}} \right| \right.} \\ \left. {{site}{is}{reference}} \right) \end{matrix}} \right)}}{{LOD}_{N} = {\log_{10}\left( \frac{\begin{matrix} {P\left( \left. {{observed}{data}{in}{normal}} \right| \right.} \\ \left. {{site}{is}{reference}} \right) \end{matrix}}{\begin{matrix} {P\left( \left. {{observed}{data}{in}{normal}} \right| \right.} \\ \left. {{site}{is}{mutated}} \right) \end{matrix}} \right)}}$

MuTect expects somatic mutations to occur at a rate of ˜1 in a Mb and requires LOD_(T)>=6.3. MuTect requires that a mutation not be in dbSNP and have a LOD_(N)>=2.3 since non-dbSNPs are expected to occur at a rate of 100 per Mb. Both cutoffs are chosen to guarantee a false positive rate less than half of the expected somatic mutation rate. Finally, additional post-processing steps are performed, including testing that the alternate allele is observed in both read directions. MuTect requires at least 14 tumor reads and 8 normal reads for a mutation to be considered.

SomaticIndelDetector (SID).

For a given mutation site, SID considers candidate indels using counts-based thresholding and an indel consensus voting scheme. The indel with the largest number of supporting reads, or votes, is chosen as the putative indel call. This call is reported if there is:

Enough coverage (default: normal >=4 reads, tumor >=6 reads),

A large fraction of reads at that site support the putative call (default: >=30%)

This fraction is sufficiently large compared to those supporting any site of the indel (default: >=70%)

Indel calls in a tumor sample are annotated as “Germline” if there is even weak evidence for the same indel in the normal sample; otherwise, they are labeled “Somatic.” Calls only observed in the normal samples are ignored. SID takes BAM files as input and outputs VCF and BED formatted putative calls.

Mutation Filtering.

The callers output all candidate mutation calls, including germline mutations and other calls with low statistical confidence. We filtered the mutation caller output to only somatic mutations, mutations designated “KEEP” by MuTect and mutations occurring within the CDS of RefSeq Genes. The tables below detail the specific filters applied to MuTect and SomaticIndelDetector output:

TABLE 38 MuTect Filter Description tumor_f > 0.1 At least 10% of the tumor reads must be variant t_alt_sum/t_alt_count > Average quality of the variant base calls > 28 28 t_alt_count − Conservatively require at least 3 reads where variant not in Q0 map_Q0_reads − or in improperly paired reads. improper_pairs >= 3 t_alt_count > 10 * When MuTect allows one variant normal read, require at least n_alt_count 10 variant tumor reads. dbsnp_site NE ‘DBSNP’ Ignore variants present in dbSNP v132 SomaticIndelDetector Filter Description T_STRAND_COUNTS_C[12]/ At least 10% of the tumor variant reads must be on each (. . ._C1 + . . ._C2) > 0.1 strand T_AV_MAPQ_C > 28 Average quality of the variant calls > 28

REFERENCES

Cibulskis, K. et al. Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nat Biotechnology (2013).doi:10.1038/nbt.2514

MuTect: www.broadinstitute.org/cancer/cga/mutect

SID: gatkforums.broadinstitute.org/discussion/35/somatic-indel-detection

TABLE 16 Druggability status for Table 2 genes/fusions Pre- Table registration 16 (pre- Gene Approved approval) Phase III Phase II Phase I preclinical TOP1 belotecan N cositecan; gimatecan; irinotecan, camptothecin hydrochloride; irinotecan, camptothecin, liposomal, (Aphios); irinotecan HyACT; Calando; Yakult; HM- irinotecan hydrochloride; irinotecan, irinotecan 30181A; (BioAlliance); topotecan PharmaEngine; HCl + namitecan; cisplatin + etirinotecan floxuridine, camptothecin irinotecan pegol Celator; prodrug, (Celator); firtecan Mersana; APH-0804; pegol; TLC- labetuzumab- irinotecan 388 SN-38; Genz- (Champions); hydrochloride; 644282; SER-203; SN- hRS7- simmitecan 38; topotecan + SN-38; hydrochloride vincristine irinotecan prodrug (LipoCure); bead, topotecan Biocompatibles (EnduRx Pharmaceuticals) SRD5A1 dutasteride N idronoxil N N N VIM N N N pritumumab N N IGFBP2 N N N N N OGX-225 SPP1 N N N N N N MDK N N N N N CAMI-103; CMS-101 MUC16 N N oregovomab N DMUC-5754A N RET sorafenib; vandetanib; apatinib motesanib N JNJ-26483327 MG-516; sunitinib malate; diphosphate; NMS-173; cabozantinib; SAR- RET kinase regorafenib 302503 inhibitor (Bionomic) MAP2K2 trametinib N ARRY- selumetinib; PD-0325901; N 438162 refametinib; ARRY-704; pimasertib; TAK-733; WX-554 GDC-0623; BI-847325; AS-703988 MAPK1 N N N N N AEZS-129; AEZS-136; AEZS-134; SCH-722984; SCH-772984 BRAF pazopanib; N N RAF-265; ARQ-761; AB-024; b-raf vemurafenib; XL-281; ARQ-736 inhibitors dabrafenib LGX-818 (Sareum); BRAF kinase inhibitor (Selexagen Therapeutics); BeiGene-283; DP-4978; TL- 241 MUC16 N N oregovomab N DMUC-5754A N MET cabozantinib; N tivantinib; MGCD- AMG-208; X-379; crizotinib rilotumumab; 265; TAS-115; metatinib; onartuzumab; foretinib; volitinib; PRS-110; ficlatuzumab; SAR-125844; ASP-08001; BMS- S-49076 ARGX-111; 777607; DCC-2701; golvatinib; DCC-2721; INCB- MG-516; AL- 028060; 2846; CG- LY- 206481; T- 2875358 1840383; cMet-EGFR dual inhibitors (CrystalGenomics); bispecific antibodies (Hoffmann-La Roche) PTK2 N N N PF- GSK-2256098; CFAK-C4; 04554878 BI-853520; FAK inhibitor VS-4718 (Verastem); CTX- 0294945; CTx-0294886; FAK inhibitors (Takeda) ACE* alacepril; benazepril; N perindopril + N amlodipine + N delapril + manidipine indapamide + enalapril (Chiesi); captopril; amlodipine maleate captopril + HCTZ; (Servier) (GlaxoSmithKline) captopril slow release (Sankyo); cilazapril; delapril; delapril + indapamide (Chiesi); diltiazem, Alza; enalapril maleate; enalapril maleate + HCTZ; enalapril + nitrendipine; enalapril (KRKA); enalaprilat; felodipine + enalapril; fosinopril; imidapril; lisinopril; lisinopril + HCTZ; moexipril; perindopril; quinapril hydrochloride; quinaprilat; ramipril; felodipine + ramipril; perindopril + indapamide, Serv; saralasin acetate; spirapril; temocapril; trandolapril; zofenopril; trandolapril + verapamil, Aven; lercanidipine + enalapril (Recordati); zofenopril + HCTZ; piretanide + ramipril; benazepril + HCTZ; amlodipine + benazepril; moexipril + HCTZ; amlodipine + perindopril, Servier; ASA + atorvastatin + ramipril + metoprolol ER (Zydus Cadila); ramipril + hydrochlorothiazide; (S)-amlodipine + ramipril (Emcure); quinapril/hydrochlorothiazide ADAM9 N N N N N N CDK6 N N palbociclib alvocidib; LEE-011 N LY- 2835219 IKBKB N N N N N EC-70124 RARA tamibarotene N N IRX-5183 N N LYN dasatinib N nintedanib bafetinib JNJ-26483327 Bcr-Abl/Lyn inhibitor (AB Science) NTRK3 N N N N N PLX-7486 ERBB2 trastuzumab; trastuzumab, neratinib; lapuleucel- Her-VAXX; Lovaxin B; trastuzumab Enhanze XL-647; T; AVX- VM-206; TH-1 emtansine; dacomitinib; 901; AE-37; ARRY-380; (Algeta); pertuzumab; lapatinib nelipepimut- BMS- JNJ-26483327; trastuzumab- ditosylate; S; 690514; S-222611; antibody catumaxomab; afatinib trastuzumab MVA-BN- doxorubicin conjugates (Celltrion, HER2; (Merrimack); (Synthon); Biocad, varlitinib; cipatinib; CUDC-101; Biocon, MM-111; TrasGEX; Her-2/neu Synthon, AC-480; trastuzumab Stradobody Harvest ovarian (Hanwha (Gliknik); Moon, cancer Chemical); ARX-788; Aryogen) vaccine trastuzumab Etbx-021; SN- (Generex); (Pfizer); IDN- 34003; IBI- margetuximab; 6439 302; NT-004; poziotinib; ICT-140; PR-610 ONS-1050; Sym-013; anti-HER2 X anti-CD3 (Emergent Biosolutions); Z-650; breast cancer vaccine (Cel-Sci); JNJ- 28871063; trastuzumab (PlantForm, BioXpress, biOasis Technologies, Stada, Natco, Curaxys, Oncobiologics, Alteogen, Mabion) RHOA N N N N N N RB1 N N N N SGT-RB94 N THRA N N N N N N CBL N N N N N N ALK crizotinib N N AP-26113; X-396; ASP- NMS-E628; RG-7853; 3026 aurora LDK-378; kinase + ALK TSR-011; inhibitor NMS-E628 (Sareum, AstraZeneca); ALK inhibitors (AstraZeneca, Cephalon, Aurigene); ARN-5032; DLX-521

TABLE 17 Druggability status for Table 3 genes/fusions Pre- registration (pre- Gene approved approval) Phase III Phase II Phase I preclinical ESR1 estramustine N acolbifene TAS-108; icaritin; SR-16388; phosphate estradiol ARN-810 VAL-201; sodium; GTx-758; SERM + ethinyl endoxifen; toxin estradiol afimoxifene (SEEK); sulfonate; estradiol fulvestrant; (BHR raloxifene Pharma); hydrochloride; NDC-1407; tamoxifen; anticancer toremifene MAb citrate; (Shenogen) trilostane; RPS6KB1 N N N N AZD-5363; p70S6 AT-13148; kinase LY- inhibitors S6KAKT1 (Sentinel)

TABLE 19 Gene Fusions Table 19 5′ gene 3′ gene Druggable Cancer Type symbol symbol gene Prostate Adenocarcinoma ABCD3 DPYD DPYD Sarcoma ACTG2 ALK ALK Lung Adenocarcinoma ADAMTS16 TERT TERT Brain Lower Grade Glioma ATRX BCL2 BCL2 Gastric Adenocarcinoma B4GALT1 RAF1 RAF1 Gastric Adenocarcinoma BRD3 LCN2 BRD3 Gastric Adenocarcinoma CASZ1 MTOR MTOR Acute Myeloid Leukemia CHD1 MTOR MTOR Uterine Corpus Endometrioid Carcinoma CPA6 PTK2 PTK2 Breast invasive carcinoma DAB1 IL12RB2 IL12RB2 Lung Adenocarcinoma DDI2 MTOR MTOR Sarcoma FRS2 MDM2 MDM2 Sarcoma GLIS3 TERT TERT Lung Adenocarcinoma HIF1A PRKCH HIF1A Breast invasive carcinoma HPRT1 CTPS2 HPRT1 Breast invasive carcinoma IL12RB2 DAB1 IL12RB2 Breast invasive carcinoma IL6R C1orf112 IL6R Breast invasive carcinoma KCMF1 PRKDC PRKDC Lung Adenocarcinoma KIF5B MET MET Breast invasive carcinoma MAPK14 EFHA1 MAPK14 Sarcoma MDM2 SPATS2 MDM2 Thyroid carcinoma MTMR12 TERT TERT Bladder Urothelial Carcinoma NOTCH2 EIF2B3 NOTCH2 Sarcoma NTRK1 DYNC2H1 NTRK1 Kidney renal clear cell carcinoma PDCD6 TERT TERT Lung Adenocarcinoma PHKB PDE3A PDE3A Uterine Carcinosarcoma RARA SLC9A3R1 RARA Liver hepatocellular carcinoma SLC12A7 TERT TERT Sarcoma SMARCA4 EEF2 EEF2 Breast invasive carcinoma STARD13 TNFRSF8 TNFRSF8 Lung Adenocarcinoma TICAM1 IL12RB1 IL12RB1 Sarcoma TRIO TERT TERT Prostate Adenocarcinoma TRPM8 UGT1A9 TRPM8 Sarcoma TSPAN3 MDM2 MDM2 Breast invasive carcinoma TTLL7 TERT TERT Brain Lower Grade Glioma USP46 PDGFRA PDGFRA Gastric Adenocarcinoma WNK2 BRD3 BRD3 Cervical squamous cell carcinoma and endocervical ZNF226 AKT2 AKT2 adenocarcinoma

TABLE 20 Breakpoints for Gene Fusions from Table 19 Table TCGA 20 Tumor 5′ 3′ Fusion Cancer Sample Gene 5′ 5′ 5′ Gene 3′ 3′ 3′ Breakpoint SEQ Name Type Barcode Symbol Accession Chromosome Breakpoint Symbol Accession Chromosome Breakpoint Sequence ID NO PDCD6| Clear TCGA- PDCD6 10016 chr5 272852 TERT 7015 chr5 1282548 TTCCTGTGGAACGTT 200 TERT Cell BP- TTCCAGAGGGTCGA Renal 4991- TAAAGACAGGAGTG Cell 01A- GAGTGAT|ATCAGA Carcinoma 01R- CAGCACTTGAAGAG 1334- GGTGCAGCTGCGGG 07 AGCTGTCGGAAGCA GA TSPAN3| Sarcoma TCGA- TSPAN3 10099 chr15 77344775 MDM2 4193 chr12 69202269 ACCTCTATGCTGAGG 201 MDM2 DX- GGTGTGAGGCTCTA A23R- GTAGTGAAGAAGCT 01A- ACAAGAA|CAGGCA 11R- AATGTGCAATACCA A26T- ACATGTCTGTACCTA 07 CTGATGGTGCTGTAA SLC12A7| Hepatocellular TCGA- SLC12A7 10723 chr5 1111983 TERT 7015 chr5 1282739 CGGAGGCTCCGGGC 202 TERT Carcinoma BC- ACCCCCGAGGGCCC A3KG- CGAGCCCGAGCGCC 01A- CCAGCCCG|GGGGT 11R- TGGCTGTGTTCCGGC A213- CGCAGAGCACCGTC 07 TGCGTGAGGAGATC CT FRS2| Sarcoma TCGA- FRS2 10818 chr12 69864310 MDM2 4193 chr12 69202988 GTGGTTACAGCACC 203 MDM2 DX- ATCAGTAGGTACAG A3M1- ACATGTTGGTATTGC 01A- ACATTTG|CCGTCCG 11R- CCCAGGTGCTGAGA A22K- GGGAGCAGGGCGC 07 GGGTCGGCGGGCGC GA CHD1| Acute TCGA- CHD1 1105 chr5 98199112 MTOR 2475 chr1 11273623 GAATGTCTAAAAGA 204 MTOR Myeloid AB- GTATACAAATCCTGA Leukemia 2939- ACAAATTAAGCAAT 03A- GGAGAAA|GAATTC 01T- TGGGTCATGAACAC 0740- CTCAATTCAGAGCAC 13 GATCATTCTTCTCAT CHD1| Acute TCGA- CHD1 1105 chr5 98204199 MTOR 2475 chr1 11273623 TTCCCATTTCTGAAG 205 MTOR Myeloid AB- AATCTGAAGAGCTG Leukemia 2939- GATCAGAAGACATT 03A- CAGCATT|GAATTCT 01T- GGGTCATGAACACC 0740- TCAATTCAGAGCAC 13 GATCATTCTTCTCAT CHD1| Acute TCGA- CHD1 1105 chr5 98199112 MTOR 2475 chr1 11273623 AATGAGAAGAATGA 206 MTOR Myeloid AB- TCGTGCTCTGAATTG Leukemia 2939- AGGTGTTCATGACCC 03A- AGAATT|CTTTCTCC 01T- ATTGCTTAATTTGTT 0740- CAGGATTTGTATACT 13 CTTTTAGACATT MAPK14| Invasive TCGA- MAPK14 1432 chr6 36044379 EFHA1 221154 chr13 22113824 GGGATGCATAATGG 207 EFHA1 Breast AO- CCGAGCTGTTGACT Carcinoma A129- GGAAGAACATTGTT 01A- TCCTGGTA|AAACTT 21R- CAGTCAAGAAGCTG A10J-07 ACAAAAAAGGACAT CGAGGATACACTGT CA TICAM1| Lung TCGA- TICAM1 148022 chr19 4831636 IL12RB1 3594 chr19 18180463 GTCCTGGCCCACAG 208 IL12RB1 Adeno 05- GCTGCCATTCAATGC carcinoma 4426- AATACGTCATGCTCT 01A- GAGCCC|GGGCTGC 01R- CGGCTGCGCCACTG 1206- GGTCCTGGGGTCCT 07 GGGGGCTGGGGCTTC TICAM1| Lung TCGA- TICAM1 148022 chr19 4831630 IL12RB1 3594 chr19 18182962 CCACTGGTTCTGTGT 209 IL12RB1 Adeno 05- GGGTGTCGGCAGGA carcinoma 4426- ATGTGCCACGTCTG 01A- GTTCAGG|GATCCG 01R- GGGCTGCCGGCTGC 1206- GCCACTGGGTCCTG 07 GGGTCCTGGGGGCT GG DAB1|IL12RB2 Invasive TCGA- DAB1 1600 chr1 57611102 IL12RB2 3595 chr1 67845789 CCCTTCACCTTTAAA 210 Breast AN- CCTCTTTATCAAAGT Carcinoma A0AM- GGCTTCACTGCGATC 01A- CTGAC|GGGAATTTT 11R- GTCTGCAAGGTGAG A034- AGGCAGTGTTAAGG 07 ATGATGAGTCCAC IL12RB2| Invasive TCGA- IL12RB2 3595 chr1 67845806 DAB1 1600 chr1 57611102 CTGCTGGTGAAAGT 211 DAB1 Breast AN- TCCCACGGAAATGA Carcinoma A0AM- GAGGGAATTTTGTCT 01A- GCAAGGT|CAGGAT 11R- CGCAGTGAAGCCAC A034- TTTGATAAAGAGGTT 07 TAAAGGTGAAGGGGT IL12RB2| Invasive TCGA- IL12RB2 3595 chr1 67845733 DAB1 1600 chr1 57611052 TCTCCCAAAATTCAC 212 DAB1 Breast AN- ATCCAATAAACAGCC Carcinoma A0AM- TGCAGCCCCGAGTG 01A- ACATAT|GTCCGGTA 11R- CAAAGCCAAATTGA A034- TCGGGATTGATGAA 07 GTTTCCGCAGCTCG GLIS3| Sarcoma TCGA- GLIS3 169792 chr9 4117768 TERT 7015 chr5 1282739 CTGCTGATCCACATG 213 TERT DX- AGAGTCCACTCTGG A3LS- GGAGAAGCCCAACA 01A- AGTGTAC|GGGGTT 11R- GGCTGTGTTCCGGC A21T- CGCAGAGCACCGTC 07 TGCGTGAGGAGATC CT ADAMTS16|TERT Lung TCGA- ADAMTS16 170690 chr5 5191903 TERT 7015 chr5 1282739 GATACAGGTCTTGG 214 Adeno 44- ACTGGCCTTCACCAT carcinoma 2662- TGCCCATGAGTCTG 01A- GACACAA|GGGTTG 01R- GCTGTGTTCCGGCC 0946- GCAGAGCACCGTCT 07 GCGTGAGGAGATCC TG ABCD3| Prostate TCGA- ABCD3 5825 chr1 94956803 DPYD 1806 chr1 97981497 CTTTAGCAACGCCAA 215 DPYD Adeno CH- ATGGAGATGTTTTG carcinoma 5764- ATCCGAGACCTTAAT 01A- TTTGAA|TCACAATA 21R- TGGAGCTTCCGTTTC 1580- TGCCAAGCCTGAACT 07 ACCCCTCTTTTA SMARCA4| Sarcoma TCGA- SMARCA4 6597 chr19 11151982 EEF2 1938 chr19 3983208 TCTGCCGGACCTCCT 216 EEF2 K1- CTTCGATCTCCTCCA A3PO- GCGTGCCCTCCTCGA 01A- TGGCC|CAACCTCAT 11R- TGACTCCCCCGGGC A21T- ATGTCGACTTCTCCT 07 CGGAGGTGACTG ZNF226| Cervical TCGA- ZNF226 7769 chr19 44669953 AKT2 208 chr19 40748529 ATTCAGCCCTGACTT 217 AKT2 Squamous IR- CTCAAAAAGCACTG Cell A3LH- CACAGAGGAGGAG Carcinoma 01A- GCAGCAGA|ACCCC 21R- ATGGACTACAAGTG A213- TGGCTCCCCCAGTGA 07 CTCCTCCACGACTGAG ZNF226| Cervical TCGA- ZNF226 7769 chr19 44669953 AKT2 208 chr19 40748529 AATTCTCCCTGACTT 218 AKT2 Squamous IR- CTCAAAAAGCACTG Cell A3LH- CACAGAGGAGGAG Carcinoma 01A- GCAGCAGA|ACCCC 21R- ATGGACTACAAGTG A213- TGGCTCCCCCAGTGA 07 CTCCTCCACGACTGAG ACTG2| Sarcoma TCGA- ACTG2 72 chr2 74128558 ALK 238 chr2 29446380 GAGATGATGCCCCC 219 ALK IW- CGGGCTGTCTTCCCC A3M6- TCCATTGTGGGCCGC 01A- CCTCGC|CACCAGGA 11R- GCTGCAAGCCATGC A21T- AGATGGAGCTGCAG 07 AGCCCTGAGTACAA ACTG2| Sarcoma TCGA- ACTG2 72 chr2 74128564 ALK 238 chr2 29449940 ATGCCCCCCGGGCT 220 ALK IW- GTCTTCCCCTCCATT A3M6- GTGGGCCGCCCTCG 01A- CCACCAG|TGATGG 11R- AAGGCCACGGGGAA A21T- GTGAATATTAAGCAT 07 TATCTAAACTGCAGT ACTG2| Sarcoma TCGA- ACTG2 72 chr2 74128564 ALK 238 chr2 29449940 TGATGCCCCCCGGG 221 ALK IW- CTGTCTTCCCCTCCA A3M5- TTGTGGGCCGCCCTC 01A- GCCACC|AGTGATG 22R- GAAGGCCACGGGGA A21T- AGTGAATATTAAGC 07 ATTATCTAAACTGCA CASZ1| Gastric TCGA- CASZ1 54897 chr1 10765549 MTOR 2475 chr1 11288975 ATGAAGTGACACCC 222 MTOR Adeno BR- CCAGCTACATCCGA carcinoma 8590- GGAGGTTCTAGGAC 01A- CTGCTACG|AGCTGA 11R- CTATAGCACTAGTGA 2402- AATGCTGGTCAACAT 13 GGGAAACTTGCCTC DDI2| Lung TCGA- DDI2 84301 chr1 15944303 MTOR 2475 chr1 11227574 ATTCTAACACTCCGG 223 MTOR Adeno MP- CCGCTGCCTCCGGCT carcinoma A4SW- GCTGTAGCTTATTAT 01A- TAATG|CTGGCTCTC 21R- GGCTGCGGGGATGC A24X- CAGACTCGAGCTCG 07 CACAGCGCGCGGA B4GALT1| Gastric TCGA- B4GALT1 2683 chr9 33166756 RAF1 5894 chr3 12641914 CTGGACAGGGCTGA 224 RAF1 Adeno HU- AGGTGAGGCTGATT carcinoma A4GH- CGCTGTGACTTCGAA 01A- TTGCATC|CAAGCAG 11R- CGGGGACTCCTCAG A24K- GGCAGGCGGGCAGC 31 GACAGTGCGGTGGTG HIF1A| Lung TCGA- HIF1A 3091 chr14 62207906 PRKCH 5583 chr14 61995793 AAAAATCTCATCCAA 225 PRKCH Adeno 44- GAAGCCCTAACGTG carcinoma 2668- TTATCTGTCGCTTTG 01A- AGTCAA|AGAGATCT 01R- GAAACTGGACAATG 0946- TCCTGTTGGACCACG 07 AGGGTCACTGTAA HIF1A| Lung TCGA- HIF1A 3091 chr14 62207766 PRKCH 5583 chr14 61995805 CGAAGTCTGCCAGTT 226 PRKCH Adeno 44- TACAGTGACCCTCGT carcinoma 2668- GGTCCAACAGGACA 01A- TTGTCC|AGTTTCTTT 01R- ATGTATGTGGGTAG 0946- GAGATGGAGATGCA 07 ATCAATATTTTAA HPRT1| Invasive TCGA- HPRT1 3251 chrX 133627542 CTPS2 56474 chrX 16657355 GATGATCTCTCAACT 227 CTPS2 Breast AR- TTAACTGGAAAGTCT Carcinoma A24W- AGGTTGTTGGCAGA 01A- AGATAT|GCCCGAG 11R- CACAACCCTGGCAAT A169- TTGGGAGGAACAAT 07 GAGACTGGGAATAA HPRT1| Invasive TCGA- HPRT1 3251 chrX 133609340 CTPS2 56474 chrX 16685822 ATAAATTCTTTGCTG 228 CTPS2 Breast AR- ACCTGCTGGATTACA Carcinoma A24W- TCAAAGCACTGAAT 01A- AGAAAT|AGTGATA 11R- GAGTTTGCAAGAAA A169- CTGCCTTAACTTGAA 07 AGATGCTGATTCCA HPRT1| Invasive TCGA- HPRT1 3251 chrX 133609375 CTPS2 56474 chrX 16638444 GCACTGAATAGAAA 229 CTPS2 Breast AR- TAGTGATAGATCCAT Carcinoma A24W- TCCTATGACTGTAGA 01A- TTTTAT|GGTGATGT 11R- TCCTTTTATAGAAGA A169- AAGACACAGACATC 07 GGTTCGAGGTAAA HPRT1| Invasive TCGA- HPRT1 3251 chrX 133627542 CTPS2 56474 chrX 16657355 GATGATCTCTCAACT 230 CTPS2 Breast AR- TTAACTGGAAAGAA Carcinoma A24W- TGTCTTGATTGTGGA 01A- AGATAT|GCCCGAG 11R- CACAACCCTGGCAAT A169- TTGGGAGGAACAAT 07 GAGACTGGGAATAA HPRT1| Invasive TCGA- HPRT1 3251 chrX 133609363 CTPS2 56474 chrX 16685820 GATTACATCAAAGC 231 CTPS2 Breast AR- ACTGAATAGAAATA Carcinoma A24W- GTGATAGATCCATTC 01A- CTATGAC|TGATAGA 11R- GTTTGCAAGAAACT A169- GCCTTAACTTGAAAG 07 ATGCTGATTCCACA IL6R|C1orf112 Invasive TCGA- IL6R 3570 chr1 154420647 C1orf112 55732 chr1 169790820 GGACAGAATCCAGG 232 Breast E9- AGTCCTCCAGCTGA Carcinoma A1RF- GAACGAGGTGTCCA 01A- CCCCCATG|CAGGAT 11R- AATGCTGACTACAG A157- ATTATTTCAGAAAAC 07 ACTCAAATTGTGTCG KIF5B| Lung TCGA- KIF5B 3799 chr10 32304500 MET 4233 chr7 116411617 CCAACTCACCCAAGT 233 MET Adenocarcinoma 93- GCAATTCGTGGAGG A4JN- AGGTGCATTTGTTCA 01A- GAACAG|AGGATTG 11R- ATTGCTGGTGTTGTC A24X- TCAATATCAACAGCA 07 CTGTTATTACTAC KIF5B| Lung TCGA- KIF5B 3799 chr10 32306145 MET 4233 chr7 116411932 GCACTGAAAGAAGC 234 MET Adenocarcinoma 93- TAAAGAAAATGCAT A4JN- CTCGTGATCGCAAAC 01A- GCTATCA|GCAAGA 11R- GTACACACTCCTCAT A24X- TTGGATAGGCTTGTA 07 AGTGCCCGAAGTGT BRD3| Gastric TCGA- BRD3 8019 chr9 136917428 LCN2 3934 chr9 130912517 GTATGCAGGACTTC 235 LCN2 Adenocarcinoma HU- AACACCATGTTTACA A4H2- AATTGTTACATTTAT 01A- AACAAG|TTCCAGG 11R- GGAAGTGGTATGTG A251- GTAGGCCTGGCAGG 31 GAATGCAATTCTCAG MDM2| Sarcoma TCGA- MDM2 4193 chr12 69233549 SPATS2 65244 chr12 49883267 CATTGTCCATGGCAA 236 SPATS2 DX- AACAGGACATCTTAT A1KZ- GGCCTGCTTTACATG 01A- TGCAA|TAGTTCCTA 11R- ATAAGAGCAACAAT A24X- GAAATTATCCTGGTT 07 TTGCAGCACTTT NOTCH2| Bladder TCGA- NOTCH2 4853 chr1 120458963 EIF2B3 8891 chr1 45392411 CATGCCTACTAGCCT 237 EIF2B3 Urothelial FD- CCCTAACCTTGCCAA Carcinoma A5BS- GGAGGCAAAGGATG 01A- CCAAGG|TGGAGCA 21R- GCGTGACTTCATTGG A26T- AGTGGACAGCACAG 07 GAAAGAGGCTGCTC NTRK1| Sarcoma TCGA- NTRK1 4914 chr1 156851401 DYNC2H1 79659 chr11 103306708 AACGCCACAGCATC 238 DYNC2H1 DX- AAGGATGTGCACGC A3LY- CCGGCTGCAAGCCC 01B- TGGCCCAG|AAGAT 11R- CCCTTACAATACCTG A27Q- AGAGGTCTTGTTGCC 07 CGTGCCCTTGCAATA PHKB| Lung TCGA- PHKB 5257 chr16 47723028 PDE3A 5139 chr12 20799464 ACTTCAGATCCGTGG 239 PDE3A Adenocarcinoma MN- CGGAGACAAGCCAG A4N5- CCTTGGACTTGTATC 01A- AGCTGT|TTGGTATC 11R- TTACTACACAGCCTA A24X- TTCCAGGCCTCTCAA 07 CTGTGATTAATG USP46| Lower TCGA- USP46 64854 chr4 53522650 PDGFRA 5156 chr4 55143576 GTGGAAGCAACCAC 240 PDGFRA Grade CS- TAATATAAACACCTC Glioma 6665- CCATGTATAGGAAG 01A- GCTGGAG|CGTTTG 11R- GGAAGGTGGTTGAA 1896- GGAACAGCCTATGG 07 ATTAAGCCGGTCCCAA USP46| Lower TCGA- USP46 64854 chr4 53494288 PDGFRA 5156 chr4 55140771 GGTCAATTTTGGAA 241 PDGFRA Grade CS- ACACATGCTACTGTA Glioma 6665- ACTCCGTGCTTCAGG 01A- CATTGT|CCTGGTTG 11R- TCATTTGGAAACAG 1896- AAACCGAGGTATGA 07 AATTCGCTGGAGGG MTMR12| Thyroid TCGA- MTMR12 54545 chr5 32263219 TERT 7015 chr5 1282739 ACATGAAGTACAAA 242 TERT Gland BJ- GCAGTGAGTGTCAA Carcinoma A4O9- CGAAGGCTATAAAG 01A- TCTGTGAG|AGGGG 11R- TTGGCTGTGTTCCGG A250- CCGCAGAGCACCGT 07 CTGCGTGAGGAGAT CC ATRX| Lower TCGA- ATRX 546 chrX 77041468 BCL2 596 chr18 60795992 AATCAAACAGAGGC 243 BCL2 Grade DB- CGCATGCTGGGGCC Glioma A4XF- GTACAGTTCCACAAA 01A- GGCATCC|TCATGGG 11R- CTCAGCGGTCATGTT A27Q- TTCGCTTGAACGCCT 07 TGTCGGCTTCTGT TRPM8| Prostate TCGA- TRPM8 79054 chr2 234894509 UGT1A9 54600 chr2 234675680 CATGTTATCCACCAA 244 UGT1A9 Adenocarcinoma CH- CATCCTGCTGGTCAA 5766- CCTGCTGGTCGCCAT 01A- GTTTG|GGAATTTGA 11R- AGCCTACATTAATGC 1580- TTCTGGAGAACATG 07 GAATTGTGGTTT KCMF1| Invasive TCGA- KCMF1 56888 chr2 85262227 PRKDC 5591 chr8 48772278 CACAGTCTTTTACTT 245 PRKDC Breast EW- GTCCCTATTGTGGAA Carcinoma A1P4- AAATGGGCTATACG 01A- GAGACA|GTACCCT 21R- GAGTGAGGAAATGA A144- GTCAATTTGATTTCT 07 CAACCGGAGTTCAG CPA6| Endometrial TCGA- CPA6 57094 chr8 68536411 PTK2 5747 chr8 141774389 AAACAGAAGAGGAA 246 PTK2 Endometrioid A5- GCATATGCACTGAA Adenocarcinoma A0G5- GAAAATATCCTATCA 01A- ACTTAAG|AAACAG 11R- ATGATTATGCTGAG A040- ATTATAGATGAAGA 07 AGATACTTACACCATG RARA| Carcinosarcoma TCGA- RARA 5914 chr17 38508759 SLC9A3R1 9368 chr17 72758151 ACCATCGCCGACCA 247 SLC9A3R1 N8- GATCACCCTCCTCAA A4PQ- GGCTGCCTGCCTGG 01A- ACATCCT|GCGCGAG 11R- CTTCGGCCTCGGCTC A28V- TGTACCATGAAGAA 07 GGGCCCCAGTGGCT WNK2| Gastric TCGA- WNK2 65268 chr9 95947892 BRD3 8019 chr9 136910543 ACAAGGGGCTGGAC 248 BRD3 Adenocarcinoma HU- ACGGAGACCTGGGT A4H2- GGAGGTGGCCTGGT 01A- GTGAGCTG|CAGAG 11R- GAAGATGGATGGCC A251- GAGAGTACCCAGAC 31 GCACAGGGCTTTGC TGC TRIO|TERT Sarcoma TCGA- TRIO 7204 chr5 14420130 TERT 7015 chr5 1282739 ATCGCCCACTCCAGA 249 DX- AGTAGCATGGAAAT A1L3- GGAGGGCATCTTCA 01A- ACCACAA|AGGGGT 11R- TGGCTGTGTTCCGGC A24X- CGCAGAGCACCGTC 07 TGCGTGAGGAGATCC TTLL7| Invasive TCGA- TTLL7 79739 chr1 84464614 TERT 7015 chr5 1282739 CCGCTTGCAGCGGG 250 TERT Breast C8- GACGCGAGGACCCG Carcinoma A131- GGCTGGGCTTTCCTC 01A- ACCCGGG|GGTTGG 11R- CTGTGTTCCGGCCGC A115- AGAGCACCGTCTGC 07 GTGAGGAGATCCTGG STARD13| Invasive TCGA- STARD13 90627 chr13 33859649 TNFRSF8 943 chr1 12164568 CTCACAGACCGTGTT 251 TNFRSF8 Breast BH- CTTCTGCGCCGTGCC Carcinoma A0C7- TGGGAACTTGACAA 01B- TCATCC|GGCTCATC 11R- CTGTAAGGAGAGCG A115- TCTTGTAGTCTGATC 07 AAATCGCAAGTAC

TABLE 21 Druggability Status for Table 19 Genes/Fusions Pre- registration Table 21 (pre- Gene approved approval) Phase III Phase II Phase I Preclinical AKT2 N N N N ARQ- RX-1792; NT-113; 092; TAS-117 BAY- 1125976 ALK crizotinib N N AP-26113; X-396; NMS-E628; aurora RG-7853; ASP- kinase + ALK inhibitor LDK-378; 3026; (Sareum, TSR-011; AstraZeneca); ALK NMS-E628 inhibitors (AstraZeneca, Cephalon, Aurigene); ARN-5032; DLX-521 BCL2 N N N PBI-1402; N VAL-101; BP-100- PNT-2258; R- 1.02; sabutoclax (—)-gossypol; navitoclax; RG-7601 BRD3 N N N N Y-803 N DPYD N N N eniluracil TAS-114 N EEF2 denileukin N moxetumomab cintredekin N Glioblast-13 diftitox pasudotox besudotox FGFR3 ponatinib asitinib lenvatinib dovitinib JNJ- N lactate; 4275649 ENMD-2076; 3; BGJ- AZD-4547 398; LY- 2874455; S-49076 HIF1A camptothecin, 2- RX-0047; ATSP- Calando methoxy 9172; ATSP-9172; P- estradiol; 3971 SPC- 2968 HPRT1 Butocin N N N N N IL12RB1 N N N INXN- AS-1409; N 2001/1001; NHS- IL-12 IL12 IL12RB2 N N N IL-12 NHS-IL- N 12; AS- 1409 IL6R tocilizumab N ARRY-438162 givinostat; L-6 inhibitors, ALX-0061 Interprotein; IL-6 antagonists, Protagonist Therapeutics; APX- 007 MAPK14 pirfenidone N N ralimetinib ARRY- N 614; thioureidobutyronitrile MDM2 N N N N SAR- p53-mdm2/mdm4 405838; dual inhibitors, RG- Adamed; PXN-527; 7388; ATSP-7041; MDM2 RO- inhibitors, Amgen 5503781; CGM- 097; DS- 3032 MET cabozantinib; N tivantinib; MGCD-265; AMG- X-379; metatinib; crizotinib rilotumumab; foretinib; 208; PRS-110; ASP- onartuzumab; ficlatuzumab; TAS-115; 08001; ARGX-111; BMS-777607; volitinib; DCC-2701; DCC- golvatinib; SAR- 2721; MG-516; AL- INCB-028060; 125844; 2846; CG-206481; T- LY-2875358; S-49076 1840383; cMet- apitolisib EGFR dual inhibitors (CrystalGenomics); bispecific antibodies (Hoffmann-La Roche) MTOR everolimus; ridaforolimus N quinacrine; P-7170; nPT-MTOR; SB2343; temsirolimus XL-765; CBLC- STP-503; X-480; dactolisib; 137, INK- ABTL-0812; X-414; PKI-587; PF- 128, CC214; HMPL-518; 04691502; AZD- PQR-309; PQR-401; CC-223 2014; mTOR inhibitor/PI3 CC-115; kinase inhibitor, Lilly- PWT- 1; PIM/PI3k/mTOR 33957; inhibitors, Inflection DS-7423; Biosciences GDC- 0084; DS-3078; LY- 3023414; PI3 kinase/mTOR inhibitor, Lilly NOTCH2 N N N OMP-59R5 N N NTRK1 N N N milciclib N tyrosine kinase maleate inhibitors (Bristol- Myers Squibb); PLX- 7486 PDE3A amrinone; N N parogrelil CR-3465 CLC-2001 anagrelide hydrochloride; hydrochloride; K-134; RPL- enoximone; 554; cilostazol; cilostazol, loprinone Genovate hydrochloride; loprinone hydrochloride; loprinone hydrochloride PDGFRA imatinib nintedanib orantinib; ENMD-2076; N DCC-2618; CG- mesilate; motesanib; olaratumab; 206481 pazopanib; linifanib X-82; sunitinib, crenolanib; dasatinib; nilotinib; regorafenib PRKDC N N vosaroxin N SF-1126, N Dbait; CC-115 PTK2 N N N defactinib GSK- CFAK-C4; FAK 2256098; inhibitor, Verastem; CEP- CTX-0294945; x- 37440; 0294886 BI- 853520; VS-4718 RAF1 sorafenib N N iCo-007; XL- RO- BIB-024; STP503; 281 5126766; DP-4978; HM-95573; MLN- TAK-632 2480 RARA tamibarotene N N IRX-5183 N N TERT N N GV-1001 VX-001; GX- TeloB- telomerase vaccine, 301- Vax Geron; hTERT DNA vaccine, Inovio TNFRSF8 brentuximab N N AFM-13; N N vedotin XmAb-2513 TRPM8 N N N N D-3263 N

TABLE 22 Cancer Types Newly Associated with Gene Fusions Druggable Cancer Type Gene A Gene B Orientation (5′/3′) gene Cancer type precedent Papillary renal cell FGFR3 TACC3 FGFR3/TACC3 FGFR3 Bladder cancer; carcinoma Squamous cell lung cancer; Glioblastoma; Head & Neck squamous cell carcinoma; Cervical sqaumous cell carcinoma; Low grade glioma Squamous cell Lung SEC16A NOTCH1 SEC16A/NOTCH1 NOTCH1 Breast Cancer; Carcinoma Thyroid Gland Carcinoma

TABLE 23 Breakpoints of Gene Fusions from Table 22 TCGA Tumor 3′ Table 23 Cancer Sample 5′ Gene 5′ Gene 3′ 3′ Breakpoint Fusion Name Type Barcode Symbol 5′ Accession Chromosome 5′ Breakpoint Symbol 3′ Accession Chromosome Breakpoint Sequence SEQ ID NO FGFR3| Papillary TCGA- FGFR3 2261 chr4 1808661 TACC3 10460 chr4 1741429 TCCTCACA 252 TACC3 Renal A4- CCTGCTCC Cell 7287- TCAGCTCC Carcinoma 01A- CGGTTCTC 11R- CTCCTGTG 2139-07 TCGCCTTT AC|GTCGG TGGACGTC ACGGTAAG GACACGGT CCAGGTCC TCCACCAG CTGCT FGFR3| Papillary TCGA- FGFR3 2261 chr4 1808633 TACC3 10460 chr4 1741500 GCCGCGCC 253 TACC3 Renal A4- CTCCCAGA Cell 7287- GGCCCACC Carcinoma 01A- TTCAAGCA 11R- GCTGGTGG 2139-07 AGGACCTG GA|ACTGG GGAAGATC ATGGACAG GTTCGAAG AGGTTGTG TACCAGGC CATGG FGFR3| Papillary TCGA- FGFR3 2261 chr4 1808661 TACC3 10460 chr4 1741429 AGCAGCTG 254 TACC3 Renal A4- GTGGAGG Cell 7287- ACCTGGAC Carcinoma 01A- CGTGTCCT 11R- TACCGTGA 2139-07 CGTCCACC GAC|GTAA AGGCGACA CAGGAGG AGAACCGG GAGCTGAG GAGCAGGT GTGAGGA FGFR3| Papillary TCGA- FGFR3 2261 chr4 1808637 TACC3 10460 chr4 1742650 CGCCCTCC 255 TACC3 Renal A4- CAGAGGCC Cell 7287- CACCTTCA Carcinoma 01A- AGCAGCTG 11R- GTGGAGG 2139-07 ACCTGGAC CGT|GTCC TTCTCCGA CCTCTTCA AGCGTTTT GAGAAACA GAAAGAG GTGATCG FGFR3| Papillary TCGA- FGFR3 2261 chr4 1808561 TACC3 10460 chr4 1741689 GAGGGCC 256 TACC3 Renal A4- ACCGCATG Cell 7287- GACAAGCC Carcinoma 01A- CGCCAACT 11R- GCACACAC 2139-07 GACCTGTA CAT|GATC ATGGACAG GTTCGAAG AGGTTGTG TACCAGGC CATGGAGG AAGTTC SEC16A| Squamous TCGA- SEC16A 9919 chr9 139352036 NOTCH1 4851 chr9 139418396 GTACGCCC 257 NOTCH1 Cell NC- AGTCCCTG Lung A5HK- GGTGCCGA Carcinoma 01A- GACCTGCC 11R- CCCTGCCT A26W- AGTTTCCA 07 GG|ACCCC AACCCGTG CCTCAGCA CCCCCTGC AAGAACGC CGGGACAT GCCAC

TABLE 24 Druggability Status of Genes/Fusions of Table 22 Pre-registration Gene approved (pre-approval) Phase III Phase II Phase I preclinical FGFR3 ponatinib masitinib lenvatinib dovitinib lactate; JNJ-42756493; N ENMD-2076; BGJ-398; LY- AZD-4547 2874455; S-49076 NOTCH1 N N N N OMP-52M51 Debio-0826; TR-4; Notch antibody (AVEO); Notch1 inhibitors (Interprotein); BMS871; NTR-4

TABLE 39 Total Genes No. no. of (Entrez Druggable Cancer Event type Q positive patients Cytoband ID) genes KM evidence Endometrial Fusion 2.18E−03 5 258 11p15.5, RPLP2 poor outcome Endometrioid 4p13 (6181), Adenocarcinoma ATP8A1 (609542) Cervical Fusion 3.56E−03 5 54 17q21.2 KRT15 poor outcome Squamous (3866), Cell KRT19 Carcinoma (3880) Colorectal Loss of 9.69E−03 4 105 11q22-q23 ATM poor outcome Adenocarcinoma: Function (472) KRAS Mutation Mutation Ductal Fusion 1.46E−02 7 265 17p11.2, USP22 poor outcome Breast 17p13 (23326), Carcinoma: MYH10 ER (160776) Positive and HER2 Negative Endometrial In-Peak 3.40E−02 8 171 3q26.2 MECOM poor outcome Endometrioid Gene (2122) Adenocarcinoma: Amplification Microsatellite Stable Endometrial Loss of 5.04E−02 4 188 16p13.3 CREBBP poor outcome Endometrioid Function (1387) Adenocarcinoma Mutation Cutaneous Gain of 6.69E−02 5 214 7q34 PRSS37 poor outcome Melanoma Function (136242) Mutation Endometrial In-Peak 7.52E−02 4 94 8p11.2 FKSG2 poor outcome Serous Gene (59347) Adenocarcinoma Deletion Cutaneous Gain of 7.94E−02 5 214 6p21.3 STK19 poor outcome Melanoma Function (8859) Mutation Endometrial Loss of 8.05E−02 30 38 17p13.1 TP53 TP53 favorable Serous Function (7157) outcome Adenocarcinoma: Mutation Microsatellite Stable Colorectal In-Peak 8.58E−02 4 45 13q12.3 CDX2 poor outcome Adenocarcinoma: Gene (1045) KRAS Amplification Mutation, Stage 3 or 4 Colorectal Loss of 8.77E−02 4 105 18q21.1 SMAD4 poor outcome Adenocarcinoma: Function (4089) KRAS Mutation Mutation Colorectal Gain of 9.10E−02 10 21 12p12.1 KRAS KRAS poor outcome Adenocarcinoma: Function (3845) (pre- Microsatellite Mutation clinical) Stable

Example 7 Identification of Status of TP53

Advances in both molecular diagnostics and the understanding of cancer biology are raising the bar for clinical trial paradigms with the expectation that more effective patient stratification will improve outcome and expedite approval of effective cancer drugs.

Mutational status of TP53 has been identified as a predictive biomarker of treatment response and prognosis. For example, TP53 wild-type (WT) patients have been shown to exhibit significantly increased progression-free survival following therapies including adjuvant 5-fluorouracil and cetuximab combination treatments compared to patients harboring TP53 mutations.

TP53 mutation annotations were obtained from ONCOMINE™ NGS Mutation Browser (Compendia Biosciences, MI). In total 776 patients were assessed for TP53 mutation status; 259 patients contained at least one mutation in TP53 and were annotated as TP53 mutant while 519 patients lacked a detected TP53 mutation and were annotated as TP53 wild type. TP53 wild type and TP53 mutant annotations were then mapped at the patient level to corresponding microarray samples from the TCGA breast dataset. When mutation annotations were mapped to patients with corresponding microarray data, 327 patients were annotated as TP53 wild type and 188 were annotated as TP53 mutant. TP53 wild type and TP53 mutation signatures were generated from a differential expression analysis of the TCGA breast datasets. Gene lists were ranked by p-value according to Student's two class t-test. Genes differentially upregulated in TP53 wild type patients contributed to the TP53 wild type signature whereas genes that were upregulated in TP53 mutant patients contributed to the TP53 mutant signature. Each signature contained the top 1% of ranked genes (n=204). All genes in the TP53 wild type and TP53 mutation signature were highly significant after correcting for false discovery (Q<0.0001). The Q-value was calculated as (p-value/p-value rank)*number of genes measured.

Five ONCOMINE™ cancer types contained sufficient TP53 mutation status data to complete an analysis. Of these, significantly increased signature expression was found in TP53 WT compared to TP53 mutated clinical samples from breast (p<0.001; n=189 WT, 37 mutant), lung (p=0.0003; n=23 WT, 18 mutated), liver (p=0.0069; n=74 WT, 11 mutated) and ovarian (p=0.05; n=22 WT, 15 mutated) cancer patients and a trend was found within lymphoma patients (p=0.068; n=65 WT, 16 mutated) (see FIGS. 5-7 and 9-10 ). Table 40 contains the TP53 WT TCGA breast cancer signature.

The clinically-derived expression signature effectively distinguishes TP53 WT from mutant tumor samples.

TABLE 40 TP53 WT Signature Genes SUSD3 BAG1 ZNF214 USP30 CEP120 DMXL1 ERBB4 SLC24A1 MKL2 CA12 P4HTM PCP2 AGBL2 SYTL4 SLC7A2 KIF12 C1orf64 NME5 HEXIM2 ANKHD1-EIF4EBP3 ACBD4 TMEM161B RERG BRD8 EIF4EBP3 FSIP1 SLC16A6 VEZF1 LOC644189 TMEM128 CAMLG MLPH ZNF484 PJA2 HVCN1 FAM47E LRBA FBXO38 TCEAL5 TCTN1 C14orf25 EXOC6 LOC100129623 CHIC1 TOX4 USP47 FAM174A WFS1 RNF135 SEPSECS POLK C14orf19 TRIM4 LOC646976 KIAA1370 SPG11 TCEAL3 SLC7A8 XPC RG9MTD2 TLE3 CCNH ZC3H6 MED13L CELSR1 GLIPR1L2 ANXA9 SFRS12 CXXC5 TBC1D9B PCBD2 TTC8 LOC100131801 C9orf68 TCEAL4 TCEAL6 GAMT CACNA1D KCTD3 MAN2B2 ABCC8 ANKRD42 OBFC1 CST5 CRY2 LOC440459 MRFAP1L1 SCAMP1 LRRC48 PCM1 GMPR2 PTGER3 ZNF24 C7orf63 DDB2 CST3 TMEM101 RHBDD1 TIGD6 PTPRT NDFIP1 WDFY3 KIAA0232 RAI2 CHCHD5 REEP5 TMEM26 GREB1 KCNE4 FUT8 PCDH19 CCDC103 PGR ZFYVE1

Example 8

In accordance with methods of the Example 9, 8,690 clinical samples were analyzed for mutation status and 7,202 clinical samples were analyzed for fusion status.

Novel mutations and fusion events were identified. Table 44 shows novel mutations identified in CDNK2A and ALK. Table 41-43 identifies novel fusion events identified in the analysis.

The druggability of each target and available clinical trials are also provided in the Tables.

The novel mutations and fusion events provide an opportunity not only for further development for target treatments but also potential options for future targets of chemotherapeutics.

Example 9

In accordance with methods of previous Examples 1-8, 8,690 clinical samples were analyzed for mutation status and 7,202 clinical samples were analyzed for mutation status. Novel mutation and cancer association events were identified. Tables 45 and 46 show the novel mutations/cancer associations.

The druggability of each target and likely outcome are also provided in Table 45.

The novel mutation/cancer association events provide an opportunity not only for further development for target treatments but also potential options for future targets of chemotherapeutics.

Example 10

In accordance with methods of previous Examples 1-8, 8,690 clinical samples were analyzed for mutation status and 7,202 clinical samples were analyzed for mutation status

Novel mutation/cancer association events are shown in Table 47.

Novel fusion events and associated cancers are shown in Table 48. breakpoints and druggability are shown in Table 49. The status of additional drugs relevant to fusion partners are shown in Table 50.

In certain embodiments, the disclosure provides:

A kit comprising a set of probes that specifically recognize at least one fusion of two genes chosen from: Table 42 or 49.

A composition comprising a set of probes that specifically recognize at least one gene fusion of two genes chosen from: Table 42 or 49.

A set of probes that specifically recognize a nucleic acid comprising at least one of SEQ ID NOs: 1-289.

An isolated nucleic acid comprising at least one sequence selected from SEQ ID NOs: 1-289.

A method of detecting bladder urothelial carcinoma, breast carcinoma, endometrial endometrioid adenocarcinoma, colon adenocarcinoma, glioblastoma multiforme, clear cell renal cell carcinoma, papillary renal cell carcinoma, acute myeloid leukemia, brain lower grade glioma, lung adenocarcinoma, ovarian serous cystadenocarcinoma, prostate adenocarcinoma, rectal cutaneous melanoma, and thyroid gland carcinoma in a sample, the method comprising: amplifying a nucleic acid comprising a sequence selected from SEQ ID NOs: 1-289; and detecting the presence of the nucleic acid comprising a sequence selected from SEQ ID NOs: 1-289; wherein detecting the nucleic acid comprising a sequence selected from SEQ ID NOs: 1-289, indicates that one or more of the above cancers is present in the sample.

A kit comprising a set of probes that specifically hybridize to at least one nucleic acid comprising a break point from Table 42 or 49.

A set of probes that specifically hybridize to at least one nucleic acid comprising a break point from Tables 42 or 49.

In the kit, the nucleic acid can be a gene fusion chosen from Table 41.

A method of detecting bladder urothelial carcinoma, breast carcinoma, endometrial endometrioid adenocarcinoma, colon adenocarcinoma, glioblastoma multiforme, clear cell renal cell carcinoma, papillary renal cell carcinoma, acute myeloid leukemia, brain lower grade glioma, lung adenocarcinoma, ovarian serous cystadenocarcinoma, prostate adenocarcinoma, rectal cutaneous melanoma, and thyroid gland carcinoma in a sample, the method comprising: amplifying a gene fusion chosen from Table 41 or 48 or a fusion gene product; and detecting the presence of the gene fusion chosen from Table 41 or 48 or fusion gene product in the sample; wherein detecting the presence of the gene fusion chosen from Table 41, indicates that one or more of the above cancers is present in the sample.

An isolated gene fusion nucleic acid of between 100 and 10,000 nucleotides in length and comprising at least 25 nucleotides on either side of one of the break points in Table 42 or 49.

The isolated gene fusion nucleic acid can have a gene fusion partner that is two genes chosen from: TPM1 and ALK, PRKAR1A and ALK, NCOA1 and ALK, LPP and CASR, MDM2 and EGFR, FGFR3 and ELAVL3, B2M and GNAS, DOCK8 and JAK2, HNF1B and NOTCH1, NFASC and NTRK1, SSBP2 and NTRK1, SQSTM1 and NTRK1, TBL1XR1 and PIK3CA, AKAP13 and RET, FKBP15 and RET, TBL1XR1 and RET, CEP85L and ROS1, CLCN6 and RAF1, TRAK1 and RAF1, PRKACA and AKT1, PRKACA and AKT2, MLL and FYN, and TTC13 and JAK2, SEC16A and NOTCH1, ERC1 and RET, GTF2IRD1 and ALK, HTATSF1 and BRS3, CDH1 and CCDC132, CCDC132 and CDH1, ERBB2 and SLC29A3, MET and TFG; TFG and MET, NOTCH2 and MNDA, IRF2BP2 and NTRK1, EIF2C2 and PTK2, RARA and HOXB3, STAT3 and ETV4,and GFAP and VIM, VIM and GFAP, TOP1 and C17orf64, and TP53 and KIAA0753.

In the above method the sample is a patient sample.

The above method can further comprise diagnosing the patient as having bladder urothelial carcinoma, breast carcinoma, endometrial endometrioid adenocarcinoma, colon adenocarcinoma, glioblastoma multiforme, clear cell renal cell carcinoma, papillary renal cell carcinoma, acute myeloid leukemia, brain lower grade glioma, lung adenocarcinoma, ovarian serous cystadenocarcinoma, prostate adenocarcinoma, rectal cutaneous melanoma, or thyroid gland carcinoma when a nucleic acid comprising a sequence selected from SEQ ID NOs: 1-289 is present in the patient sample.

A kit comprising a set of probes, wherein the set of probes specifically recognize a gene variant chosen from Table 41, Table 44, Table 45, Table 46, Table 47 or a combination thereof.

A composition comprising a set of probes that specifically recognize a gene variant from Table 41, Table 44, Table 45, Table 46, Table 47 or a combination thereof.

A set of probes that specifically recognize a gene variant from Table 41, Table 44, Table 45, Table 46, Table 47 or a combination thereof.

A method of detecting bladder carcinoma, breast carcinoma, cervical cell carcinoma, colon adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, clear cell renal cell carcinoma, kidney renal papillary cell carcinoma, acute myeloid leukemia, brain lower grade glioma, liver hepatocellular carcinoma, lung adenocarcinoma, squamous cell lung carcinoma, ovarian serous adenocarcinoma, prostate adenocarcinoma, rectal adenocarcinoma, cutaneous melanoma, stomach adenocarcinoma, thyroid carcinoma, or uterine corpus endometrioid carcinoma, the method comprising: amplifying a nucleic acid comprising a variant selected from Table 7 and/or Table 11; and detecting the presence of the variant selected from Table 41, Table 44, Table 45, Table 46, Table 47 or a combination thereof; wherein detecting the variant selected from Table 41, Table 44, Table 45, Table 46, Table 47 or a combination thereof, indicates one ore of the above cancers is present in the sample.

A kit comprising a set of probes that specifically hybridize to a variant selected from Table 41, Table 44, Table 45, Table 46, Table 47 or a combination thereof.

A set of probes that specifically hybridize to a gene variant selected from Table 41, Table 44, Table 45, Table 46, Table 47 or a combination thereof.

The method of claim 17, wherein the sample is a patient sample.

The method of claim 20, further comprising diagnosing the patient as having bladder carcinoma, breast carcinoma, cervical cell carcinoma, colon adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, clear cell renal cell carcinoma, kidney renal papillary cell carcinoma, acute myeloid leukemia, brain lower grade glioma, liver hepatocellular carcinoma, lung adenocarcinoma, squamous cell lung carcinoma, ovarian serous adenocarcinoma, prostate adenocarcinoma, rectal adenocarcinoma, cutaneous melanoma, stomach adenocarcinoma, thyroid carcinoma, or uterine corpus endometrioid carcinoma when a variant selected from Table 41, Table 44, Table 45, Table 46, Table 47 or a combination thereof is present in the patient sample.

An isolated genetic construct comprising an antisense polynucleotide sequence of a sequence selected from SEQ ID NOs:1-289.

The isolated genetic construct can further comprise a promoter operatively linked to the antisense polynucleotide.

The isolated genetic construct can be a vector. The vector can be a viral vector

A method comprising contacting a cell comprising a gene fusion with a drug, wherein the gene fusion is disclosed in Table 41 or Table 49 and the drug is one or more of the drugs in Table 43 or Table 49.

A method comprising administering to a patient with a gene fusion a drug, wherein the gene fusion is disclosed in Table 41 or Table 49.

The method of claim 27, wherein the patient is diagnosed with cancer.

The method of claim 28, wherein the cancer is a cancer type disclosed in Table 41 or Table 49.

A method comprising contacting a nucleic acid sample from a patient with a reaction mixture comprising two primers, wherein a first primer is complementary to one gene and a second primer is complementary to a second gene, wherein the fusion of the first gene and the second gene is detectable by the presence of an amplicon generated by the first primer and the second primer, wherein the fusion breakpoint is one of the breakpoints of Table 42 or 49, and wherein a patient with an amplicon is administered one or more of the drugs in Table 43 or Table 49.

A method comprising performing a bioassay to detect one or more gene fusions in a sample from a subject, wherein at least one of the gene fusions is selected from those in Table 42 or Table 49, receiving the results of the bioassay into a computer system, processing the results to determine an output, presenting the output on a readable medium, wherein the output identifies therapeutic options recommended for the subject based on the presence or absence of the gene fusions.

In the above method, the bioassay can include probes specific for one or more of the breakpoints of Table 42 or Table 49.

A kit, comprising: a set of probes, wherein each probe specifically hybridizes to a nucleic acid comprising a breakpoint from Table 42 or Table 49.

In the kit, each non-naturally occurring probe can comprise: a nucleic acid sequence configured to specifically hybridize to the nucleic acid comprising the breakpoint from Table 42 or Table 49, and a detectable moiety covalently bonded to the nucleic acid sequence.

In the kit, each non-naturally occurring probe can comprise: a deoxyribonucleic acid sequence comprising at least one deoxyuridine (dU) residue in place of a deoxythymidine residue.

A method, comprising: amplifying a nucleic acid comprising at least one gene fusion from Table 41 or Table 49 from a sample; and detecting the presence of the at least one gene fusion by at least one of: contacting the composition with at least one probe, wherein each probe specifically hybridizes to the nucleic acid, or observing the presence of a non-natural or non-native chemical structure in the nucleic acid; wherein detecting the presence of the at least one gene fusion indicates that at least one cancer from Table 41 or Table 49 is present in the sample.

A system, comprising: a nucleic acid amplifier configured to amplify a nucleic acid comprising at least one gene fusion from Table 41 or Table 49 from a sample, to yield an amplified nucleic acid; a detector configured to detect the presence of the at least one gene fusion in the amplified nucleic acid by at least one of (i) contacting the composition with at least one probe, wherein each probe specifically hybridizes to the nucleic acid, or (ii) observing the presence of a non-natural or non-native chemical structure in the nucleic acid, and further configured to transmit a detection indication; and a computer system configured to receive the detection indication and determine that at least one cancer from Table 41 or Table 49 is present in the sample, based on the detection indication.

A non-transitory computer readable program storage unit encoded with instructions that, when executed by a computer, perform a method, comprising: receiving an input comprising at least a cancer type and an event type, wherein the cancer type is selected from Table 44, 45, 46 and/or 47 and the event type is selected from Table 44, 45, 46 and/or 47; querying a database for at least one entry comprising a plurality of fields, wherein the plurality of fields comprises at least one of the cancer type and the event type; transmitting an output comprising at least one field of the plurality from the at least one entry, wherein the at least one field comprises at least one gene, at least one druggable gene, at least one drug targeting the at least one druggable gene, or a prognosis.

A method, comprising: administering to a patient having at least one gene fusion selected from the gene fusions listed in Table 41 at least one drug selected from the drugs listed in Table 43.

A method, comprising: contacting a nucleic acid sample from a patient with a reaction mixture comprising a first primer complementary to a first gene and a second primer complementary to a second gene, wherein a fusion of the first gene and the second gene is detectable by the presence of an amplicon generated by the first primer and the second primer, wherein the fusion comprises a breakpoint selected from the breakpoints listed in Table 42 or Table 49.

In the above method, at least one drug can be selected from the drugs listed in Table 43, 45, 46 and/or 47 is administered to the patient, in response to a detection of the fusion.

A non-transitory computer readable program storage unit encoded with instructions that, when executed by a computer, perform a method, comprising: receiving RNA sequence data from at least one cancer cell line; running at least one gene fusion caller on the sequence data, to identify possible breakpoints between fused genes in the processed data; filtering said possible breakpoints, to retain candidate breakpoints, wherein each candidate breakpoint is in a 5′ untranslated region (UTR) or a coding DNA sequence (CDS) of a functional gene region and each candidate breakpoint does not occur in an intron; annotating the candidate breakpoints with at least one annotation useful in determining a relevance of a gene fusion for at least one of cancer diagnosis, cancer prognosis, or cancer treatment, wherein the gene fusion comprises the candidate breakpoint.

In the non-transitory computer readable program storage unit, at least one gene fusion caller can be selected from TopHat and deFuse.

A non-transitory computer readable program storage unit encoded with instructions that, when executed by a computer, perform a method, comprising: receiving mutation data from at least one cancer cell line; annotating the mutation data with at least one of variant classification, variant position, or variant change, to yield annotated mutation data; filtering the annotated mutation data, to yield gene region mutation data; classifying the gene region mutation data as hotspot, deleterious, or other; and nominating a gene comprising the gene region mutation as a gain of function, loss of function, or recurrent other gene, based on the relative frequency of mutations in the gene and the classifications of all gene region mutations in the gene.

In the non-transitory computer readable program storage unit, the variant classification can be selected from splice site, 3′ untranslated region (UTR), 5′ UTR, intron, missense, nonsense, nonstop, silent, frame shift insertion, frame shift deletion, in-frame insertion, in-frame deletion, or non-coding exon.

In the non-transitory computer readable program storage unit, filtering the annotated mutation data can comprise excluding mutations outside of a known transcript and mutations not associated with a known gene.

In the non-transitory computer readable program storage unit, classifying the mutation as hotspot can comprise determining its presence at the same variant position in at least three cancer cell lines, wherein the variant classification is missense, nonstop, in-frame insertion, in-frame deletion, or non-coding exon.

In the non-transitory computer readable program storage unit, wherein classifying the mutation as deleterious can comprise observing the variant classification is nonsense, frame shift insertion, or frame shift deletion.

A method, comprising: detecting one or more gene fusions in a sample from a subject, to yield gene fusion detection data, wherein at least one of the gene fusions is selected from the gene fusions listed in Table 41 or 49, receiving by a computer system the gene fusion detection data, identifying by the computer system at least one therapeutic option recommended for the subject, based on the gene fusion detection data.

A system, comprising: a detector configured to (i) detect one or more gene fusions in a sample from a subject, to yield gene fusion detection data, wherein at least one of the gene fusions is selected from the gene fusions listed in Table 41 or 49 and (ii) transmit the gene fusion detection data; and a computer system configured to receive the gene fusion detection data and identify at least one therapeutic option recommended for the subject, based on the gene fusion detection data.

A gene fusion that is prepared by a process comprising: isolating an RNA molecule comprising the gene fusion; and synthesizing a complementary DNA (cDNA) molecule complementary to the isolated RNA molecule.

Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety.

It is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

Reference to sequence identifiers, such as those beginning with NM_, refer to the database accession numbers and the underlying sequences as they were found on Apr. 18, 2013.

TABLE 7 TCGA Disease Gene Symbol Entrez Gene ID Start Position Reference Allele Tumor Seq Allele Transcript Variant C READ ANXA1 301 74965099 G A NM_000700 p.R124H SKCM ANXA1 301 75775278 C T NM_000700 p.R124C UCEC ANXA1 301 75775279 G A NM_000700 p.R124H BRCA AR 367 66765161 A T NM_000044 p.Q58L HNSC AR 367 66765161 A T NM_000044 p.Q58L KIRP AR 367 66765161 A T NM_000044 p.Q58L LGG AR 367 66765161 A T NM_000044 p.Q58L LUAD AR 367 66765161 A T NM_000044 p.Q58L STAD AR 367 66765161 A T NM_000044 p.Q58L UCEC AR 367 66765161 A T NM_000044 p.Q58L LUAD ARAF 369 47426120 T A NM_001654 p.S214T LUAD ARAF 369 47426121 C T NM_001654 p.S214F SKCM ARAF 369 47426121 C T NM_001654 p.S214F PAAD ATP6V1A 523 113505224 T C NM_001690 p.L237P SKCM ATP6V1A 523 113505224 T C NM_001690 p.L237P LUAD CDK4 1019 58145430 C A NM_000075 p.R24L SKCM CDK4 1019 58145430 C A NM_000075 p.R24L SKCM CDK4 1019 58145431 G T NM_000075 p.R24S OV CHEK2 11200 27422947 C T NM_007194 p.R346H GBM CHEK2 11200 29083962 G C NM_007194 p.R519G HNSC CHEK2 11200 29083962 G C NM_007194 p.R519G KIRC CHEK2 11200 29083962 G C NM_007194 p.R519G PAAD CHEK2 11200 29083962 G C NM_007194 p.R519G HNSC CHEK2 11200 29091840 T C NM_007194 p.K373E KIRC CHEK2 11200 29091840 T C NM_007194 p.K373E LUAD CHEK2 11200 29091840 T C NM_007194 p.K373E SKCM CHEK2 11200 29091840 T C NM_007194 p.K373E BRCA CHEK2 11200 29092948 G A NM_007194 p.R346C LUSC CHEK2 11200 29092948 G C NM_007194 p.R346G HNSC CSNK2A1 1457 470440 T C NM_001895 p.H236R LUAD CSNK2A1 1457 470440 T C NM_001895 p.H236R LUSC CSNK2A1 1457 470440 T C NM_001895 p.H236R STAD CSNK2A1 1457 470440 T C NM_001895 p.H236R THCA CSNK2A1 1457 470440 T C NM_001895 p.H236R GBM DRD5 1816 9784478 C A NM_000798 p.S275R HNSC DRD5 1816 9784478 C A NM_000798 p.S275R LUSC DRD5 1816 9784478 C A NM_000798 p.S275R STAD DRD5 1816 9784478 C A NM_000798 p.S275R COAD ERBB3 2065 54765121 G A NM_001982 p.V104M COAD ERBB3 2065 54765121 G A NM_001982 p.V104M COAD ERBB3 2065 54765121 G T NM_001982 p.V104L READ ERBB3 2065 54765121 G A NM_001982 p.V104M CESC ERBB3 2065 56478854 6 A NM_001982 p.V104M STAD ERBB3 2065 56478854 G T NM_001982 p.V104L STAD ERBB3 2065 55478854 G A NM_001982 p.V104M UCEC ERBB3 2065 56478854 G A NM_001982 p.V104M BRCA ERBB3 2065 56482341 G T NM_001982 p.D297Y UCEC ERBB3 2065 56482341 G T NM_001982 p.D297Y UCEC ERBB3 2065 56482341 G A NM_001982 p.D297N UCEC ERBB3 2065 56482342 A T NM_001982 p.D297V HNSC FGFR3 2261 1803565 C G NM_000142 p.S249C KIRP FGFR3 2261 1803568 C G NM_000142 p.S249C LUSC FGFR3 2261 1803568 C G NM_000142 p.S249C COAD GPRC5A 9052 12952538 G A NM_003979 p.V30I UCEC GPRC5A 9052 13061271 G A NM_003979 p.V30I LUAD GPX1 2876 49395482 G C NM_000581 p.P77R SKCM GPX1 2876 49395482 G C NM_000581 p.P77R STAD GPX1 2876 49395482 G C NM_000581 p.P77R KIRC HSD17B7 51478 162769603 G A NM_016371 p.S173N PAAD HSD17B7 51478 162759603 G A NM_016371 p.S173N BRCA JUN 3725 59248409 C T NM_002228 p.E112K LUSC JUN 3725 59248409 C T NM_002228 p.E112K LUSC JUN 3725 59248409 C G NM_002228 p.E112Q COAD KDR 3791 55650977 C T NM_002253 p.R1032Q SKCM KDR 3791 55955863 G A NM_002253 p.S1100F SKCM KDR 3791 55956220 C T NM_002253 p.R1032Q LAML KIT 3815 55294077 G T NM_000222 p.D816Y LAML KIT 3815 55294078 A T NM_000222 p.D816V SKCM LHCGR 3973 48915500 C T NM_000233 p.R479Q UCEC LHCGR 3973 48915500 C A NM_000233 p.R479L SKCM LHCGR 3973 48936151 C T NM_000233 p.E206K HNSC MAP2K2 5605 4117549 A C NM_030662 p.F57L SKCM MAP2K2 5605 4117551 A C NM_030662 p.F57V STAD MAP2K2 5605 4117551 A C NM_030662 p.F57V CESC MAPK1 5594 22127164 C T NM_002745 p.E322K HNSC MAPK1 5594 22127164 C T NM_002745 p.E322K COAD MMP15 4324 56631345 G A NM_002428 p.R169H SKCM MMP15 4324 58073843 C T NM_002428 p.R169C LUAD MMP15 4324 58073844 G A NM_002428 p.R169H OV MMP3 4314 102215174 G A NM_002422 p.R316C GBM MMP3 4314 102709963 C T NM_002422 p.R316H GBM MMP3 4314 102709964 G A NM_002422 p.R316C LUAD MMP3 4314 102709964 G A NM_002422 p.R316C COAD MTOR 2475 11107160 G T NM_004958 p.S2215Y KIRC MTOR 2475 11184573 G T NM_004958 p.S2215Y KIRP MTOR 2475 11184573 G T NM_004958 p.S2215Y UCEC MTOR 2475 11184573 G T NM_004958 p.S2215Y KIRC MTOR 2475 11189545 G C NM_004958 p.F1888L UCEC MTOR 2475 11189845 G T NM_004958 p.F1888L UCEC MTOR 2475 11189847 A C NM_004958 p.F1888V OV MTOR 2475 11195525 C T NM_004958 p.A1105T KIRC MTOR 2475 11217230 C T NM_004958 p.C1483Y KIRC MTOR 2475 11217230 C A NM_004958 p.C1483F GBM MTOR 2475 11217231 A G NM_004958 p.C1483R SKCM MTOR 2475 11272938 C T NM_004958 p.A1105T GBM PIK3CB 5291 138374244 T G NM_006219 p.D1067A HNSC PIK3CB 5291 138374244 T A NM_006219 p.D1067V THCA PIK3CB 5291 138374244 T A NM_006219 p.D1067V UCEC PIK3CB 5291 138374245 C A NM_006219 p.D1067Y LUAD PIK3R2 5296 18273784 G A NM_005027 p.G373R UCEC PIK3R2 5296 18273784 G A NM_005027 p.G373R COAD POLE 5426 131760362 C A NM_006231 p.V411L COAD POLE 5426 131763257 G T NM_006231 p.P286H UCEC POLE 5426 133250289 C A NM_006231 p.V411L UCEC POLE 5426 133253184 G C NM_006231 p.P286R UCEC PPP2R1A 5518 52715971 C G NM_014225 p.P179R UCEC PPP2R1A 5518 52715982 C T NM_014225 p.R183W HNSC PPP2R1A 5518 52715983 G A NM_014225 p.R183Q STAD PPP2R1A 5518 52715983 G A NM_014225 p.R183Q UCEC PPP2R1A 5518 52716323 C T NM_014225 p.S256F UCEC PPP2R1A 5518 52716323 C A NM_014225 p.S256Y UCEC PPP2R1A 5518 52716328 C T NM_014225 p.R258C LUAD PPP2R1A 5518 52716329 G A NM_014225 p.R258H COAD PPP2R1A 5518 57407794 C T NM_014225 p.R183W COAD PPP2R1A 5518 57407794 C T NM_014225 p.R183W OV PPP2R1A 5518 57407794 C T NM_014225 p.R183W COAD PPP2R1A 5518 57408141 G A NM_014225 p.R258H HNSC PRKCA 5578 64299066 G C NM_002737 p.E33Q LUAD PRKCA 5578 64299066 G A NM_002737 p.E33K LUSC PRKCA 5578 64299066 G A NM_002737 p.E33K KIRC PRKCH 5583 61789073 C T NM_006255 p.A85V PAAD PRKCH 5583 61789073 C T NM_006255 p.A85V STAD PRKCI 5584 170013719 C A NM_002740 p.R480S COAD PRKCI 5584 171496413 C T NM_002740 p.R480C COAD PRKCI 5584 171496413 C T NM_002740 p.R480C OV PRKCI 5584 171496413 C T NM_002740 p.R480C COAD RAF1 5894 12620699 G A NM_002880 p.S257L COAD RAF1 5894 12620699 G A NM_002880 p.S257L LUAD RAF1 5894 12645699 G A NM_002880 p.S257L LUAD RAF1 5894 12645699 G C NM_002880 p.S257W SKCM RAF1 5894 12645699 G A NM_002880 p.S257L STAD RAF1 5894 12645699 G A NM_002880 p.S2S7L KIRC RHEB 6009 151188050 A T NM_005614 p.Y35N UCEC RHEB 6009 151188050 A T NM_005614 p.Y35N STAD RHOA 387 49412898 T C NM_001664 p.Y42C STAD RHOA 387 49412898 T G NM_001664 p.Y42S BRCA RHOA 387 49412905 C G NM_001664 p.E40Q HNSC RHOA 387 49412905 C G NM_001664 p.E40Q COAD SRC 6714 35464354 G C NM_005417 p.D407H OV SRC 6714 35464354 G C NM_005417 p.D407H SKCM SRCIN1 80725 36704930 C T NM_025248 p.E1045K READ SYK 6850 92676932 G T NM_003177 p.K387N LGG SYK 6850 93637110 A G NM_003177 p.K387R SKCM SYK 6850 93637110 A G NM_003177 p.K387R STAD TOP2A 7153 38552660 T C NM_001067 p.K1199E THCA TOP2A 7153 38552660 T C NM_001067 p.K1199E COAD TOP2B 7155 25643731 C T NM_001068 p.R651H UCEC TOP2B 7155 25668727 C T NM_001068 p.R651H GBM TUBA1B 10376 49523423 C T NM_006082 p.G29D STAD TUBA1B 10376 49523423 C T NM_006082 p.G29D HNSC TUBA1B 10376 49523424 C G NM_006082 p.G29R BLCA TXNRD1 7296 104725378 G A NM_003330 p.E439K CESC TXNRD1 7296 104725378 G C NM_003330 p.E439Q UCEC TXNRD1 7296 104725378 G C NM_003330 p.E439Q HNSC TXNRD1 7296 104725379 A G NM_003330 p.E439G KIRC TXNRD1 7296 104725379 A G NM_003330 p.E439G LGG VEGFB 7423 64005040 A C NM_003377 p.T187P PAAD VEGFB 7423 64005040 A C NM_003377 p.T187P HNSC VEGFB 7423 64005048 A C NM_001243733 p.T156P PAAD VEGFB 7423 64005048 A C NM_001243733 p.T156P SKCM VEGFB 7423 64005048 A C NM_001243733 p.T156P BLCA Bladder Urothelial Carcinoma BRCA Breast invasive carcinoma CESC Cervical Squamous Cell Carcinoma COAD colon adenocarcinoma GBM glioblastoma HNSC head and neck squamous cancer KIRC Kidney Renal Clear Cell Carcinoma KIRP Kidney Renal Papillary Cell Carcinoma LAML acute myeloid leukemia LGG low grade glioma LUAD lung adenocarcinoma LUSC lung squamnous cell carcinoma OV ovarian carcinoma PAAD pancreatic adenoacrcinoma READ rectal adenocarcinoma SKCM Skin Cutaneous Melanoma STAD stomach adenocarcinoma THCA thyroid carcinoma UCEC Uterine Corpus Endometrioid Carcinoma

TABLE 41 5′ gene 3′ gene Druggable Cancer Type symbol symbol gene Prostate Adenocarcinoma ACPP AXL AXL Prostate Adenocarcinoma ATM CARD18 ATM Lung Adenocarcinoma ATXN1 ARG1 ARG1 Lower Grade Glioma CLU CST3 CLU Prostate Adenocarcinoma DHX15 ETV1 none Lower Grade Glioma DOT1L TECR DOT1L Adrenal Cortex Carcinoma EXOSC10 MTOR MTOR Cervical Squamous Cell Carcinoma FAT1 NTRK3 NTRK3 Bladder Urothelial Carcinoma FHIT RUNX1 none Prostate Adenocarcinoma GUCA2A ERG none Sarcoma KANK2 ALK ALK Cutaneous Melanoma LMNA RAF1 RAF1 Prostate Adenocarcinoma MDM2 BRI3BP MDM2 Cervical Squamous Cell Carcinoma MX1 ERG none Hepatocellular Carcinoma OXR1 MET MET Papillary Renal Cell Carcinoma PLS1 PIK3CB PIK3CB Colon and Rectal Adenocarcinoma RPS6 FN1 FN1 Cutaneous Melanoma RSBN1L HGF HGF Lung Adenocarcinoma RUNX1 CEP76 none Sarcoma SEC16A RXRA RXRA Lung Adenocarcinoma STK11 HMHA1 None Ovarian Serous Cystadenocarcinoma TBL1XR1 CTNNB1 CTNNB1 Prostate Adenocarcinoma TMPRSS2 GUCA2A None Prostate Adenocarcinoma TMPRSS2 TMEM109 None Prostate Adenocarcinoma TMPRSS2 BRAF BRAF Colon and Rectal Adenocarcinoma USP7 PRKCB PRKCB Lower Grade Glioma VEGFA STK38 VEGFA

TABLE 42 5′ Gene 3′ Gene 5′ 5′ 5′ 3′ 3′ 3′ Symbol Symbol Cancer Type TCGA Tumor Sample Barcode Accession Chromosome Breakpoint Accession Chromosome Breakpoint ACPP AXL Prostate Adenocarcinoma TCGA-J4-A6G3-01A-11R-A311-07 55 chr3 132075687 558 chr19 41745606 ATM CARD18 Prostate Adenocarcinoma TCGA-KK-A6E8-01A-11R-A31N-07 472 chr11 108218092 59082 chr11 105009805 ATXN1 ARG1 Lung Adenocarcinoma TCGA-55-6978-01A-11R-1949-07 6310 chr6 16658016 383 chr6 131897847 CLU CST3 Lower Grade Glioma TCGA-CS-4944-01A-01R-1470-07 1191 chr8 27461879 1471 chr20 23618406 CLU CST3 Lower Grade Glioma TCGA-PS-A72U-01A-31R-A32Q-07 1191 chr8 27461878 1471 chr20 23614559 DHX15 ETV1 Prostate Adenocarcinoma TCGA-G9-6339-01A-12R-A311-07 1665 chr4 24585946 2115 chr7 13975521 DOT1L TECR Lower Grade Glioma TCGA-TM-A7CF-02A-11R-A32Q-07 84444 chr19 2194576 9524 chr19 14673337 DOT1L TECR Lower Grade Glioma TCGA-TM-A7CF-01A-11R-A32Q-07 84444 chr19 2194576 9524 chr19 14673337 EXOSC10 MTOR Adrenal Cortex Carcinoma TCGA-OR-A5J7-01A-11R-A29S-07 5394 chr1 11139768 2475 chr1 11190834 FAT1 NTRK3 Cervical Squamous Cell Carcinoma TCGA-HM-A3JK-01A-11R-A32Y-07 2195 chr4 187627717 4916 chr15 88680792 FHIT RUNX1 Bladder Urothelial Carcinoma TCGA-LC-A66R-01A-41R-A30C-07 2272 chr3 61237029 861 chr21 36231788 GUCA2A ERG Prostate Adenocarcinoma TCGA-KC-A4BR-01A-32R-A32Y-07 2980 chr1 42629074 2078 chr21 39817544 HMHA1 STK11 Lung Adenocarcinoma TCGA-62-8395-01A-11R-2326-07 23526 chr19 1066162 6794 chr19 1218416 STK11 HMHA1 Lung Adenocarcinoma TCGA-NJ-A4YF-01A-12R-A262-07 6794 chr19 1222005 23526 chr19 1080254 KANK2 ALK Sarcoma TCGA-FX-A48G-01A-11R-A24X-07 25959 chr19 11303507 238 chr2 29451932 LMNA RAF1 Cutaneous Melanoma TCGA-EB-A55F-01A-11R-A311-07 4000 chr1 156107492 5894 chr3 12645651 MDM2 BRI3BP Prostate Adenocarcinoma TCGA-KK-A7B2-01A-12R-A32O-07 4193 chr12 69214153 140707 chr12 125509889 MX1 ERG Cervical Squamous Cell Carcinoma TCGA-C5-A7CJ-01A-11R-A32P-07 4599 chr21 42809050 2078 chr21 39947620 OXR1 MET Hepatocellular Carcinoma TCGA-RC-A6M6-01A-11R-A32O-07 55074 chr8 107726213 4233 chr7 116411552 PLS1 PIK3CB Papillary Renal Cell Carcinoma TCGA-G7-6793-01A-11R-1965-07 5357 chr3 142315341 5291 chr3 138461531 RPS6 FN1 Colon and Rectal Adenocarcinom TCGA-AG-3609-01A-02R-0826-07 6194 chr9 19378859 2335 chr2 216259394 RSBN1L HGF Cutaneous Melanoma TCGA-EB-A5SE-01A-11R-A311-07 222194 chr7 77326339 3082 chr7 81381543 RLINX1 CEP76 Lung Adenocarcinoma TCGA-O1-A52J-01A-13R-A262-07 861 chr21 36206707 79959 chr18 12686449 SEC16A RXRA Sarcoma TCGA-DX-A3LS-01A-11R-A21T-07 9919 chr9 139352034 6256 chr9 137293495 TBL1XR1 CTNNB1 Ovarian Serous Cystadenocarcinoma TCGA-24-2280-01A-01R-1568-13 79718 chr3 176914909 1499 chr3 41274832 TMPRSS2 GUCA2A Prostate Adenocarcinoma TCGA-KC-A4BR-01A-32R-A32Y-07 7113 chr21 42879877 2980 chr1 42629281 TMPRSS2 TMEM109 Prostate Adenocarcinoma TCGA-HC-8216-01A-11R-A29R-07 7113 chr21 42860321 79073 chr11 60687158 TMPRSS2 TMEM109 Prostate Adenocarcinoma TCGA-HC-8213-01A-11R-A29R-07 7113 chr21 42852403 79073 chr11 60687197 TMPRSS2 BRAF Prostate Adenocarcinoma TCGA-FC-A6HD-01A-11R-A31N-07 7113 chr21 42866283 673 chr7 140481493 USP7 PRKCB Colon and Rectal Adenocarcinom TCGA-A6-3807-01A-01R-1022-07 7874 chr16 9057064 5579 chr16 24043457 VEGFA STK38 Lower Grade Glioma TCGA-TM-A7CF-02A-11R-A32Q-07 7422 chr6 43745395 11329 chr6 36467720 VEGFA STK38 Lower Grade Glioma TCGA-TM-A7CF-01A-llR-A32Q-07 7422 chr6 43745395 11329 chr6 36467720 5′ Gene Symbol 3′ Gene Symbol Breakpoint Sequence SEQ ID NO ACPP AXL GGTTGGCCCTGTGATCCCTCAAGACTGGTCCACGGAGTGTATGACCACAA|ACAGCCAGTCCACCAGCTGGTGAAGGAACCTTCAACTCCTGCCTTCTCGT 258 ATM CARD18 GAATATCTTGATAAATGAGCAGTCAGCAGAACTTGTACATATAGATCTAG|ACCAACTCTTGCGTAAAAAGAGAAGAATTTTTATCCATTCAGTGGGTGCA 259 ATXN1 ARG1 AAGAGAGAAAGAGTGGATTTCAGCCTGCACGGATGGTCTTGAAACACAAA|TGGTCTGCTTGAGAAACTTAAAGAACAAGAGTGTGATGTGAAGGATTATG 260 CLU CST3 ACAGAATTCATACGAGAAGGCGACGATGACCGGACTGTGTGCCGGGAGAT|CCGCCGCGCCTAGTGGGAGGCCCCATGGACGCCAGCGTGGAGGAGGAGGG 261 CLU CST3 CAGAATTCATACGAGAAGGCGACGATGACCGGACTGTGTGCCGGGAGATC|CGCCTAGGGGTCTGTACCGGGCTGGCCTGTGCCTATCACCTCTTATGCAC 262 DHX15 ETV1 GGACCTAGGGGAGGATTACCCCTCTGGCAAGAAGCGTGCGGGGACCGATG|GTGCCTATGATCAGAAGCCACAAGTGGGAATGAGGCCCTCCAACCCCCCC 263 DOM TECR AGTTCAGGAAGTGGATGAAATGGTATGGAAAAAAGCATGCAGAATACACA|GTGGAGATTCTGGACGCAAAGACAAGGGAGAAGCTGTGTTTCTTGGACAA 264 DOM TECR AGTTCAGGAAGTGGATGAAATGGTATGGAAAAAAGCATGCAGAATACACA|GTGGAGATTCTGGACGCAAAGACAAGGGAGAAGCTGTGTTTCTTGGACAA 265 EXOSC10 MTOR ATCAACGAAATGCACCTTTTAATCCAGCAGGCCCGAGAGATGCCCCTGCT|CAAGGCCTGGCATGCGTGGGCAGTGATGAACTTCGAAGCTGTGCTACACT 266 FAT1 NTRK3 TCCATTAGAGATGGCTCTGGCGTTGGTGTTTTCAAAATAGGTGAAGAGAC|AGGCAGTTGGAGCAGAACTTTTTCAACTGCAGCTGTGACATCCGCTGGAT 267 FHIT RUNX1 GTCCCTCAGGCGGCCACCCAGTGGGCACACTCCCAGGCGGCGCTCC|GGCCCCGAGAACCTCGAAGACATCGGCAGAAACTAGATGATCAGACCAAG 268 GUCA2A ERG AGGCACACTCAAACAACGACTGGTCCTCACTCACAACTGATAAGGCTTCC|CAGCCTCTGAAGTATCTCCTGGGCATTGGGCTCCTTGCAGAGAGGCTTGA 269 HMHA1 STK11 GCTGTGGCCTTCATGTCCTGTGCCCCAGAGATCTGCCCCTCCCACCCGAG|GGAAATTCAACTACTGAGGAGGTTACGGCACAAAAATGTCATCCAGCTGG 270 STK11 HMHA1 GAGTACGAACCGGCCAAGAGGTTCTCCATCCGGCAGATCCGGCAGCACAG|GTCCCCCGTCATGCGTGCCCGGAAGAGCAGCTTCAACGTGAGTGATGTGG 271 KANK2 ALK CCGGCCCAGAGCAAGGAAGTGTTATCATTCCAGCCACCTCCACCACCTGC|TGCTCCATCGCAGCTTCGCTCTGTGATGCTAATCTTCTTCACCATATGGA 272 LMNA RAF1 AAGAAGTGGCCATGCGCAAGCTGGTGCGCTCAGTGACTGTGGTTGAGGAC|ACAGCAGGATGATTGAGAATAACAACCTGAGTGCTTCTCCCAGGGCGTGG 273 MDM2 BRI3BP GGAAAATATATACCATGATCTACAGGAACTTGGTAGTAGTCAATCAGCAG|GCTGGAGCACCTGGAGAAGCAGGTCAGACTGCTCAACATCCGTCTCAACC 274 MX1 ERG TGGAGAGGCAAGGTCAGTTACCAGGACTACGAGATTGAGAMCGGATGC|TTCAGACTGTCCCGGACCCAGCAGCTCATATCAAGGAAGCCTTATCAGTT 275 OXR1 MET CTGAAACAATTGAGGATTCTAGTAATCAAGCAGCAGCCAGAGAATGGGAG|TGGAAGCAAGCAATTTCTTCAACCGTCCTTGGAAAAGTAATAGTTCAACC 276 PL51 PIK3CB TGCTCTGGTGGATATGTTTGTTTTAGCCAGTCCATCCAAGACAATCCCAC|CGCTGTCCTGCCTTCGAGAGGGCAGCTCCCACTGGAGATCCAAGTACCTG 277 RPS6 FN1 GGTCCGAATCAGTGGTGGGAACGACAAACAAGGTTTCCCCATGAAGCAGG|GAAATTCTTTGGAAGAAGTGGTCCATGCTGATCAGAGCTCCTGCACTTTT 278 RSBN1L HGF GAGGAGGCACGGTCTCGGTGGGGCCCGAGAGGCCGGCGGGGCCTCCCGGG|AGGAAAACTACTGTCGAAATCCTCGAGGGGAAGAAGGGGGACCCTGGTGT 279 RUNX1 CEP76 CCCTGAACCACTCCACTGCCTTTAACCCTCAGCCTCAGAGTCAGATGCAG|GGATGAAAATGGGATAAATAGACCAGTCTGTTCCTATGTTAAACCACTTC 280 SEC16A RXRA ACGCCCAGTCCCTGGGTGCCGAGACCTGCCCCCTGCCTAGTTTCCAGGTG|AACTCCTCCCTCACCTCCCCGACGGGGCGAGGCTCCATGGCTGCCCCCTC 281 TBL1XR1 CTNNB1 CAAGACGTTGACTTGGATCTGTCAGGTGAAGTCCTAAAGCTTGCATTCCA|CCTGGAAACGGTGGCCTCCAACGCCGCTCCCCCCTCCCGGGAATGGAGGC 282 TMPRSS2 GUCA2A AGGTCTTTGAGCTTCTTCACTGACTCCAGAGAAAAGGAGAAATTTCCATC|CTGCCGCGCCGCGCTCCTCACACCCGCTTTCACCTCCGGGCGGGGCAGGG 283 TMPRSS2 TMEM109 GTGTGATGGCGTGTCACACTGCCCCGGCGGGGAGGACGAGAATCGGTGTG|ACCCAGTCATGGCAGCCTCCAGCATCAGTTCACCATGGGGAAAGCATGTG 284 TMPRSS2 TMEM109 GACTGGAACGAGAACTACGGGCGGGCGGCCTGCAGGGACATGGGCTATAA|GAAAGCATGTGTTCAAAGCCATTCTGATGGTCCTAGTGGCCCTTATCCTC 285 TMPRSS2 BRAF CGTCGTCTGCACGCAGCCCAAATCCCCATCCGGGACAGTGTGCACCTCAA|AAAACACTTGGTAGACGGGACTCGAGTGATGATTGGGAGATTCCTGATGG 286 USP7 PRKCB GAAAGCGGGCGAGCAGCAGTTGAGCGAGCCCGAGGACATGGAGATGGAAG|GACCCCCGCAGCAAACACAAGTTTAAGATCCACACGTACTCCAGCCCCAC 287 VEGFA STK38 TGCAATGACGAGGGCCTGGAGTGTGTGCCCACTGAGGAGTCCAACATCAC|CATGCAGACCGGGTACAACAAGCTCTGTGATTGGTGGTCGCTTGGGGTGA 288 VEGFA STK38 TGCAATGACGAGGGCCTGGAGTGTGTGCCCACTGAGGAGTCCAACATCAC|CATGCAGACCGGGTACAACAAGCTCTGTGATTGGTGGTCGCTTGGGGTGA 289

TABLE 43 Pre-registration Gene Approved (pre-approval) Phase III AXL ARG1 CLU custirsen DOT1L MTOR everolimus; ridaforolimus N temsirolimus NTRK3 ALK crizotinib alectinib hydrochloride ceritinib RAF1 sorafenib N N MDM2 N N N MET cabozantinib; N tivantinib; crizotinib rilotumumab; onartuzumab; PIK3CB FN1 HGF rilotumumab RXRA bexarotene, bexarotene, gel, CTNNB1 PRKCB BRAF vemurafenib, encorafenib pazopanib, dabrafenib VEGFA bevacizumab, midostaurin, XL-647, pegaptanib bevacizumab, Biocad, octasodium, bevacizumab, Actavis, pegaptanib bevacizumab, octasodium Reliance Life Sciences Gene Phase II Phase I Preclinical AXL foretinib, BGB-324, S-49076, ningetinib TP-0903, SGI-7079, Q-4, BGB-109, CEP-40783 ARG1 PEG-arginase, Bio-Cancer CLU AB-16B5, CGEN-25008 DOT1L EPZ-01 DOT1L inhibitors, Aurigene MTOR quinacrine; XL-765; dactolisib; P-7170; CBLC-137, AZD-2014; nPT-MTOR; STP-503; X-480; ABTL-0812; X- PKI-587; PF-04691502; CC-223, CC-115; PWT-33957; DS-7423; 414; CC214; HMPL-518; PQR-309; PQR-401; apitolisib; gedatolisib, INK-128 GDC-0084; DS-3078; LY- mTOR inhibitor/PI3 kinase inhibitor, Lilly-1; PKI-587; PF-04691502; CC- 3023414; PI3 kinase/mTOR PIM/PI3k/mTOR inhibitors, Inflection 223, apitolisib, gedatolisib, INK- inhibitor, Lilly, Biosciences 128 SF-1126, SB-2343 NTRK3 TSR-011 PLX-7486 NMS-P626 ALK AP-26113; RG-7853; TSR-011; X-396; ASP-3026; CEP-37440 NMS-E628; aurora kinase + ALK inhibitor NMS-E628, PF-06463922 (Sareum, AstraZeneca); ALK inhibitors (AstraZeneca, Cephalon, Aurigene); ARN- 5032; DLX-521, TL-398, AZD-3463 RAF1 iCo-007; XL-281 RO-5126766; MLN-2480, BIB-024; STP503; DP-4978; HM-95573; TAK- pan-Raf inhibitor, Eli Lilly 632 MDM2 N DS-3032, AMG-232 p53-mdm2/mdm4 dual inhibitors, Adamed; PXN-527; ATSP-7041; MDM2 inhibitors, Amgen, DS-5272 MET MGCD-265; foretinib; AMG-208; TAS-115; X-379; metatinib; PRS-110; ASP-08001; DCC- ficlatuzumab; BMS-777607; volitinib; SAR-125844; 2721; MG-516; AL-2846; CG-206481; T- golvatinib; INCB-028060; LY- S-49076, metatinib, F-50064, 1840383; cMet-EGFR dual inhibitors( 2875358; apitolisib ARGX-111, CrystalGenomics); bispecific antibodies DCC-2701 (Hoffmann-La Roche) PIK3CB pictilisib, GS-9820, GSK- INK-1117, BAY-1082439, PQR-3xx, Piqur 2636771 SAR-260301, AZD-8186 FN1 radretumab, darleukin AS-1409 HGF HuL2G7 NK4, Kringle RXRA IRX-4310 CTNNB1 PRI-724 β-catenin inhibitors, PhaseRx, K-756, DsiRNAs, Dicerna PRKCB sotrastaurin, BRAF RAF-265, XL-281, AB-024 ARQ-761, ARQ-736, b-raf inhibitors, Sareum, B-Raf and c-RAF dual BeiGene-283 inhibitor, Redx Pharma, PLX-8394, DP-4978, TL- 241, B-Raf kinase inhibitors, Array BioPharma- 1, EN-3352, EBI-907, PI3K/BET bromodomain inhibitors, SignalRx VEGFA squalamine, PTC-299, MP-0112, ASC-06, PRS-050, VEGF vaccine, Neovacs, CEQ-300, ESBA-1008 sevacizumab, RG-7221, bevacizumab, BioXpress, bevacizumab, Natco, PAN-90806, bevacizumab, Oncobiologics, bevacizumab, TAS-115, bevacizumab, Pfizer Harvest Moon, OMP-305B83, bevacizumab, Fujifilm Kyowa Kirin Biologics, bevacizumab, LG Life Sciences, bevacizumab, Biocon, bevacizumab, Mabion, bevacizumab, Aryogen, hVEGF-trunc vaccine, Immunovo, bevacizumab, Avesthagen, bevacizumab, Alteogen, bevacizumab, Nanogen

TABLE 44 Gene Entrez Reference Tumor Seq Tumor Seq Variant Variant Variant Cancer Type Symbol Gene ID Allele Allele1 Allele2 Change Position Class Druggability Gastric Adenocarcinoma CDKN2A 1029 A C C p.V25G p.V25 Missense_Mutation Y Clear Cell Renal Cell CDKN2A 1030 A C C p.V25G p.V25 Missense_Mutation Y Carcinoma Multiple Myeloma CDKN2A 1031 A C — p.V25G p.V25 Missense_Mutation Y Prostate Adenocarcinoma CDKN2A 1032 A A C p.V25G p.V25 Missense_Mutation Y Lung Adenocarcinoma ALK 238 C G G p.W247S p.W247 Missense_Mutation Y

TABLE 45 No. Total no. Subset Event type Q positive of patients Acute Myeloid Leukemia Fusion 4.03E−02 11 168 Chromophobe Renal Cell Carcinoma Loss of Function Mutation 6.84E−02 18 65 Chromophobe Renal Cell Carcinoma Loss of Function Mutation 6.92E−02 4 65 Colorectal Adenocarcinoma: Gain of Function Mutation 3.86E−02 8 45 KRAS Mutation, Stage 3 or 4 Colorectal Adenocarcinoma: Gain of Function Mutation 9.10E−02 5 21 Microsatellite Stable Diffuse Gastric Adenocarcinoma In-Peak Gene Amplification 3.04E−02 4 52 Ductal Breast Carcinoma: Triple Negative Loss of Function Mutation 9.90E−02 51 68 Gastric Intestinal Type Adenocarcinoma In-Peak Gene Amplification 3.41E−02 4 44 Infiltrating Bladder Urothelial Carcinoma In-Peak Gene Amplification 7.16E−02 4 183 Leiomyosarcoma In-Peak Gene Deletion 1.86E−03 6 40 Lung Adenocarcinoma: EGFR Mutation Loss of Function Mutation 9.57E−03 13 21 Pancreatic Ductal Adenocarcinoma Gain of Function Mutation 8.78E−04 5 39 Pancreatic Ductal Adenocarcinoma Loss of Function Mutation 5.97E−02 5 39 Prostate Adenocarcinoma Loss of Function Mutation 1.25E−03 14 172 Colorectal Adenocarcinoma: Gain of Function Mutation 3.39E−04 10 105 KRAS Mutation Cytoband Genes (Entrez ID) Druggable genes KM evidence 17q21, 15q22 RARA (5914), PML (5371) RARA favorable outcome 17p13 TP53 (7157) TP53 poor outcome 10q23 PTEN (5728) PTEN poor outcome 7q32 IRF5 (3663) Poor outcome 22q12 NEFH (4744) poor outcome 11p13 APIP (51074) poor outcome 17p13 TP53 (7157) TP53 favorable outcome 10q26 FGFR2 (2263) FGFR2 poor outcome 20q11 E2F1 (1869) E2F1 Poor outcome 9p21 CDKN2A (1029), CDKN2B (1030) CDKN2A poor outcome 17p13 TP53 (7157) TP53 poor outcome PTCD1 (26024), ATP5J2-PTCD1 7q22 (100526740) poor outcome 4q35 DUX2 (26583) poor outcome 17p13 TP53 (7157) TP53 poor outcome 7q32 IRF5 (3663) poor outcome

TABLE 46 Subset Event type Q Acute Myeloid Leukemia Fusion 4.03E−02 Chromophobe Renal Cell Carcinoma Loss of Function Mutation 6.84E−02 Chromophobe Renal Cell Carcinoma Loss of Function Mutation 6.92E−02 Colorectal Adenocarcinoma: Gain of Function Mutation 3.86E−02 KRAS Mutation, Stage 3 or 4 Colorectal Adenocarcinoma: Gain of Function Mutation 9.10E−02 Microsatellite Stable Diffuse Gastric Adenocarcinoma In-Peak Gene Amplificatior 3.04E−02 Ductal Breast Carcinoma: Triple Negative Loss of Function Mutation 9.90E−02 Gastric Intestinal Type Adenocarcinoma In-Peak Gene Amplificatior 3.41E−02 Infiltrating Bladder Urothelial Carcinoma In-Peak Gene Amplificatior 7.16E−02 Leiomyosarcoma In-Peak Gene Deletion 1.86E−03 Lung Adenocarcinoma: EGFR Mutation Loss of Function Mutation 9.57E−03 Pancreatic Ductal Adenocarcinoma Gain of Function Mutation 8.78E−04 Pancreatic Ductal Adenocarcinoma Loss of Function Mutation 5.97E−02 Prostate Adenocarcinoma Loss of Function Mutation 1.25E−03 Colorectal Adenocarcinoma: KRAS Mutation Gain of Function Mutation 3.39E−04 Colorectal Adenocarcinoma: KRAS Mutation Gain of Function Mutation 1.53E−02 No. Total no. Subset positive of patients Cytoband Genes (Entrez ID) Acute Myeloid Leukemia 11 168 17q21, RARA (5914), 15q22 PML (5371) Chromophobe Renal Cell Carcinoma 18 65 17p13 TP53 (7157) Chromophobe Renal Cell Carcinoma 4 65 10q23 PTEN (5728) Colorectal Adenocarcinoma: 8 45 7q32 IRF5 (3663) KRAS Mutation, Stage 3 or 4 Colorectal Adenocarcinoma: 5 21 22q12 NEFH (4744) Microsatellite Stable Diffuse Gastric Adenocarcinoma 4 52 11p13 APIP (51074) Ductal Breast Carcinoma: Triple Negative 51 68 17p13 TP53 (7157) Gastric Intestinal Type Adenocarcinoma 4 44 10q26 FGFR2 (2263) Infiltrating Bladder Urothelial Carcinoma 4 183 20q11 E2F1 (1869) Leiomyosarcoma 6 40 9p21 CDKN2A (1029), CDKN2B (1030) Lung Adenocarcinoma: EGFR Mutation 13 21 17p13 TP53 (7157) PTCD1 (26024), ATP5J2-PTCD1 Pancreatic Ductal Adenocarcinoma 5 39 7q22 (100526740) Pancreatic Ductal Adenocarcinoma 5 39 4q35 DUX2 (26583) Prostate Adenocarcinoma 14 172 17p13 TP53 (7157) Colorectal Adenocarcinoma: KRAS Mutation 10 105 7q32 IRF5 (3663) Colorectal Adenocarcinoma: KRAS Mutation 4 105 5q31 PCDHA7 (56141) Druggable Subset genes KM evidence Acute Myeloid Leukemia RARA favorable outcome Chromophobe Renal Cell Carcinoma TP53 poor outcome Chromophobe Renal Cell Carcinoma PTEN poor outcome Colorectal Adenocarcinoma: Poor outcome KRAS Mutation, Stage 3 or 4 Colorectal Adenocarcinoma: poor outcome Microsatellite Stable Diffuse Gastric Adenocarcinoma poor outcome Ductal Breast Carcinoma: Triple Negative TP53 favorable outcome Gastric Intestinal Type Adenocarcinoma FGFR2 poor outcome Infiltrating Bladder Urothelial Carcinoma E2F1 Poor outcome Leiomyosarcoma CDKN2A poor outcome Lung Adenocarcinoma: EGFR Mutation TP53 poor outcome Pancreatic Ductal Adenocarcinoma poor outcome Pancreatic Ductal Adenocarcinoma poor outcome Prostate Adenocarcinoma TP53 poor outcome Colorectal Adenocarcinoma: KRAS Mutation poor outcome Colorectal Adenocarcinoma: KRAS Mutation poor outcome

TABLE 47 Cancer Gene Entrez Type Symbol Gene ID Papillary Renal Cell Carcinoma AR 367 Lobular Breast Carcinoma AR 367 Adrenal Cortex Carcinoma AR 367 Colorectal Adenocarcinoma AR 367 Pancreatic Carcinoma AR 367 Colorectal Adenocarcinoma AR 367 Pancreatic Ductal Adenocarcinoma AR 367 Prostate Carcinoma JAK1 3716 Hepatocellular Carcinoma JAK1 3716 Cancer Reference Type Allele Papillary Renal Cell Carcinoma — Lobular Breast Carcinoma — Adrenal Cortex Carcinoma GCAGCAGCAGCAGCAGCAGCAGCA (SEQ ID NO: 290) Colorectal Adenocarcinoma — Pancreatic Carcinoma GCAGCAGCA Colorectal Adenocarcinoma GCAGCAGCAGCAGCAGCAGCAGCAGCA (SEQ ID NO: 291) Pancreatic Ductal Adenocarcinoma GCAGCAGCA Prostate Carcinoma C Hepatocellular Carcinoma T Cancer Tumor Seq Type Allele 1 Papillary Renal Cell Carcinoma — Lobular Breast Carcinoma — Adrenal Cortex Carcinoma GCAGCAGCAGCAGCAGCAGCAGCA (SEQ ID NO: 290) Colorectal Adenocarcinoma — Pancreatic Carcinoma GCAGCAGCA Colorectal Adenocarcinoma GCAGCAGCAGCAGCAGCAGCAGCAGCA (SEQ ID NO: 291) Pancreatic Ductal Adenocarcinoma GCAGCAGCA Prostate Carcinoma T Hepatocellular Carcinoma T Cancer Tumor Seq Variant Variant Type Allele 2 Change position Papillary Renal Cell Carcinoma GCAGCA p.L57_in_frame_ins p.L57 Lobular Breast Carcinoma GCA p.L57_in_frame_ins p.L57 Adrenal Cortex Carcinoma — p.L57_in_frame_del p.L57 Colorectal Adenocarcinoma GCAGCA p.L57_in_frame_ins p.L57 Pancreatic Carcinoma — p.L57_in_frame_del p.L57 Colorectal Adenocarcinoma — p.L57_in_frame_del p.L57 Pancreatic Ductal Adenocarcinoma — p.L57_in_frame_del p.L57 Prostate Carcinoma — p.E188K p.E188 Hepatocellular Carcinoma C; A p.E188G; p.E188V p.E188 Cancer Variant Pan-Cancer CBI Type Class Variant Category Druggability Papillary Renal Cell Carcinoma In_Frame_Ins Hotspot Y Lobular Breast Carcinoma In_Frame_Ins Hotspot Y Adrenal Cortex Carcinoma In_Frame_Del Hotspot Y Colorectal Adenocarcinoma In_Frame_Ins Hotspot Y Pancreatic Carcinoma In_Frame_Del Hotspot Y Colorectal Adenocarcinoma In_Frame_Del Hotspot Y Pancreatic Ductal Adenocarcinoma In_Frame_Del Hotspot Y Prostate Carcinoma Missense_Mutation Hotspot Y Hepatocellular Carcinoma Missense_Mutation Hotspot Y

TABLE 48 5′ gene 3′ gene Druggable Cancer Type symbol symbol gene Esophageal Carcinoma NUP214 ABL1 ABL1 Colon and Rectal Adenocarcinoma SMEK2 ALK ALK Cutaneous Melanoma KCNQ5 ALK ALK Colon and Rectal Adenocarcinoma TRIM24 BRAF BRAF Bladder Urothelial Carcinoma CDKN2A CAPZB CDKN2A Colon and Rectal Adenocarcinoma PEX1 CDK6 CDK6 Lower Grade Glioma EGFR C7orf72 EGFR Hepatocellular Carcinoma ERBB2 PPP1R1B ERBB2 Prostate Adenocarcinoma ERBB2 PPP1R1B ERBB2 Hepatocellular Carcinoma FGFR2 BICC1 FGFR2 Lower Grade Glioma FGFR3 FBXO28 FGFR3 Prostate Adenocarcinoma FGFR3 AES FGFR3 Esophageal Carcinoma FGFR3 TACC3 FGFR3 Cervical Squamous Cell Carcinoma JAK2 RCL1 JAK2 Gastric Adenocarcinoma ZBTB17 MTOR MTOR Endometrial Endometroid KAZN MTOR MTOR Adenocarcinoma Lower Grade Glioma EPHB2 NTRK1 NTRK1 Esophageal Carcinoma RAF1 C9orf153 RAF1 Pancreatic Carcinoma RAF1 TMEM40 RAF1 Squamous Cell Lung Carcinoma CDKN2A SRGAP2 CDKN2A Hepatocellular Carcinoma SLC7A2 TERT TERT

TABLE 49 5′ Gene 3′ Gene Cancer TCGA Tumor Symbol Symbol Type Sample Barcode NUP214 ABL1 Esophageal Carcinoma TCGA-L5-A4OQ-01A-11R-A260-31 FGFR3 AES Prostate Adenocarcinoma TCGA-EJ-A7NM-01A-21R-A33R-07 FGFR3 AES Prostate Adenocarcinoma TCGA-EJ-A7NM-01A-21R-A33R-07 FGFR3 AES Prostate Adenocarcinoma TCGA-EJ-A7NM-01A-21R-A33R-07 FGFR3 AES Prostate Adenocarcinoma TCGA-EJ-A7NM-01A-21R-A33R-07 SMEK2 ALK Colon and Rectal Adenocarcinoma TCGA-F5-6864-01A-11R-1928-07 KCNQ5 ALK Cutaneous Melanoma TCGA-EB-A6QY-01A-12R-A32P-07 FGFR2 BICC1 Hepatocellular Carcinoma TCGA-ED-A7PX-01A-51R-A352-07 FGFR2 BICC1 Hepatocellular Carcinoma TCGA-ED-A7PX-01A-51R-A352-07 EGFR C7orf72 Lower Grade Glioma TCGA-E1-A7YJ-01A-11R-A34F-07 RAF1 C9orf153 Esophageal Carcinoma TCGA-L5-A4OS-01A-11R-A28J-31 CDKN2A CAPZB Bladder Urothelial Carcinoma TCGA-E7-A6MD-01A-41R-A352-07 CDKN2A CAPZB Bladder Urothelial Carcinoma TCGA-E7-A6MD-01A-41R-A352-07 CDKN2A CAPZB Bladder Urothelial Carcinoma TCGA-E7-A6MD-01A-41R-A352-07 PEX1 CDK6 Colon and Rectal Adenocarcinoma TCGA-AA-3697-01A-01R-1723-07 FGFR3 FBXO28 Lower Grade Glioma TCGA-TM-A84B-01A-11R-A36H-07 FGFR3 FBXO28 Lower Grade Glioma TCGA-TM-A84B-01A-11R-A36H-07 FGFR3 FBXO28 Lower Grade Glioma TCGA-TM-A84B-01A-11R-A36H-07 FGFR3 FBXO28 Lower Grade Glioma TCGA-TM-A84B-01A-11R-A36H-07 FGFR3 FBXO28 Lower Grade Glioma TCGA-TM-A84B-01A-11R-A36H-07 FGFR3 FBXO28 Lower Grade Glioma TCGA-TM-A84B-01A-11R-A36H-07 FGFR3 FBXO28 Lower Grade Glioma TCGA-TM-A84B-01A-11R-A36H-07 FGFR3 FBXO28 Lower Grade Glioma TCGA-TM-A84B-01A-11R-A36H-07 ZBTB17 MTOR Gastric Adenocarcinoma TCGA-BR-4357-01A-01R-1157-13 KAZN MTOR Endometrial Endometrioid Adenocarcinoma TCGA-D1-A3JQ-01A-11R-A22K-07 EPHB2 NTRK1 Lower Grade Glioma TCGA-TM-A84J-01A-11R-A36H-07 ERBB2 PPP1R1B Hepatocellular Carcinoma TCGA-KR-A7K2-01A-12R-A33R-07 ERBB2 PPP1R1B Pancreatic Adenocarcinoma TCGA-LB-A7SX-01A-11R-A33R-07 JAK2 RCL1 Cervical Squamous Cell Carcinoma TCGA-PN-A8MA-01A-11R-A36F-07 JAK2 RCL1 Cervical Squamous Cell Carcinoma TCGA-PN-A8MA-01A-11R-A36F-07 CDKN2A SRGAP2 Squamous Cell Lung Carcinoma TCGA-60-2715-01A-01R-0851-07 CDKN2A SRGAP2 Squamous Cell Lung Carcinoma TCGA-60-2715-01A-01R-0851-07 FGFR3 TACC3 Esophageal Carcinoma TCGA-LN-A5U5-01A-21R-A28J-31 SLC7A2 TERT Hepatocellular Carcinoma TCGA-DD-A73C-01A-12R-A33J-07 RAF1 TMEM40 Pancreatic Adenocarcinoma TCGA-IB-A5SS-01A-11R-A32O-07 BRAF TRIM24 Colon and Rectal Adenocarcinoma TCGA-F5-6464-01A-11R-1736-07 TRIM24 BRAF Colon and Rectal Adenocarcinoma TCGA-F5-6464-01A-11R-1736-07 5′ Gene 5′ 5′ 5′ 3′ 3′ 3′ Symbol Accession Chromosome Breakpoint Accession Chromosome Breakpoint NUP214 8021 chr9 134027281 25 chr9 133747516 FGFR3 2261 chr4 1808969 166 chr19 3061232 FGFR3 2261 chr4 1808905 166 chr19 3061233 FGFR3 2261 chr4 1808905 166 chr19 3061233 FGFR3 2261 chr4 1808661 166 chr19 3061255 SMEK2 57223 chr2 55804451 238 chr2 29940563 KCNQ5 56479 chr6 73332315 238 chr2 29498362 FGFR2 2263 chr10 123239533 80114 chr10 60461846 FGFR2 2263 chr10 123244971 80114 chr10 60461846 EGFR 1956 chr7 55268106 100130988 chr7 50143910 RAF1 5894 chr3 12627180 389766 chr9 88844544 CDKN2A 1029 chr9 21974677 832 chr1 19671746 CDKN2A 1029 chr9 21974673 832 chr1 19666067 CDKN2A 1029 chr9 21994138 832 chr1 19671746 PEX1 5189 chr7 92151518 1021 chr7 92355063 FGFR3 2261 chr4 1808661 23219 chr1 224340844 FGFR3 2261 chr4 1808989 23219 chr1 224340848 FGFR3 2261 chr4 1808645 23219 chr1 224345163 FGFR3 2261 chr4 1808645 23219 chr1 224345163 FGFR3 2261 chr4 1808586 23219 chr1 224340892 FGFR3 2261 chr4 1808591 23219 chr1 224318276 FGFR3 2261 chr4 1808661 23219 chr1 224340844 FGFR3 2261 chr4 1808582 23219 chr1 224340949 ZBTB17 7709 chr1 16269559 2475 chr1 11227574 KAZN 23254 chr1 15251068 2475 chr1 11206848 EPHB2 2048 chr1 23111569 4914 chr1 156843425 ERBB2 2064 chr17 37883777 84152 chr17 37790317 ERBB2 2064 chr17 37883798 84152 chr17 37790337 JAK2 3717 chr9 5090911 10171 chr9 4834141 JAK2 3717 chr9 5126446 10171 chr9 4860125 CDKN2A 1029 chr9 21994138 23380 chr1 206634382 CDKN2A 1029 chr9 21974677 23380 chr1 206634382 FGFR3 2261 chr4 1808661 10460 chr4 1741429 SLC7A2 6542 chr8 17354726 7015 chr5 1282731 RAF1 5894 chr3 12632350 55287 chr3 12791339 BRAF 673 chr7 140624377 8805 chr7 138255741 TRIM24 8805 chr7 138255748 673 chr7 140550012 5′ Gene Breakpoint SEQ ID Symbol Sequence NO. NUP214 CATGACTGCAGCTTCTTTCAAGAACTCTTCCACCTCCATGGTGTCCTCCT|GAAGCTGAGCTTCACTCTTGGGA 292 TCCAGTGGTCTTTTATAAAGCAAATGC FGFR3 GGACGACTCCGTGTTTGCCCACGACCTGCTGCCCCCGGCCCCACCCAGCA|ACTCAAATTCACCACCTCGGACT 293 CCTGCGACCGCATCAAAGACGAATTTC FGFR3 TGTCGGCGCCTTTCGAGCAGTACTCCCCGGGTGGCCAGGACACCCCCAGC|AACTCAAATTCACCACCTCGGAC 294 TCCTGCGACCGCATCAAAG FGFR3 TGTCGGCGCCTTTCGAGCAGTACTCCCCGGGTGGCCAGGACACCCCCAGC|AACTCAAATTCACCACCTCGGAC 295 TCCTGCGACCGCATCAAAGACGAATTT FGFR3 GCAGCTGGTGGAGGACCTGGACCGTGTCCTTACCGTGACGTCCACCGACG|GCTCCTCGCACCTACCCCAGCAA 296 CTCAAATTCACCACCTCGGACTCCTGC SMEK2 TGAAAAGGATAATATAGTTGGATCAAACAAAAACAACACAATTTGTCCCG|GTCATAGCTCCTTGGAATCACCA 297 ACAAACATGCCTTCTCCTTCTCCTGAT KCNQ5 CGATGGCTCTGGATCCTTGTCCCCACCATGCGACCATCTGCAGCCAGAAC|ACGAAAGCGTGGTAGATGAACGC 298 CCAGCCGCGGGGTCTCTCCAGCACGTT FGFR2 AGTTGGTAGAAGACTTGGATCGAATTCTCACTCTCACAACCAATGAGGAA|ACAAATACGCAGATTGCTTGGCCA 299 TCAAAACTGAAGATCGGAGCCAAATC FGFR2 AGATCTTCACTTTAGGGGGCTCGCCCTACCCAGGGATTCCCGTGGAGGAA|ACAAATACGCAGATTGCTTGGCCA 300 TCAAAACTGAAGATCGGAGCCAAATC EGFR GATCATCGAATTCTCCAAAATGGCCCGAGACCCCCAGCGCTACCTTGTCA|TTCAGGTTGGACAAGCCCTCTGAA 301 AGTTACTCCCTTACAACCTCATCATG RAF1 GAGTGGTTCTCAGCAGGTTGAACAACCTACTGGCTCTGTCCTCTGGATGG|AATTAGAGAATAAAATCCCAGCAC 302 GATGTTCCTCACTGGAGACACCAGTC CDKN2A TTGGCTATGTGTGGGGAGCAGTCACTCACAGTTTCATCCTTCTCCATCTG|GATCGGCCTCCGACCGTAACTATT 303 CGGTGCGTTGGGCAGCGCCCCCGCCT CDKN2A AGAGGTTTAGCATTGCTGCTTTCTCTTCAAAGCCTCCACCAGGTCATTCT|CCACCTGGATCGGCCTCCGACCGT 304 AACTATTCGGTGCGTTGGGCAGCGCC CDKN2A TTGGCTATGTGTGGGGAGCAGTCACTCACAGTFTCATCCTTCTCCATCTG|GTCTTCTAGGAAGCGGCTGCTGCC 305 CTAGACGCTGGCTCCTCAGTAGCATC PEX1 TGCTGCAGAATCAAGCTATAGAAGTGGTCTGGAGTCACCAGCCTGCATTC|ACACCGAGTAGTGCATCGCGATCT 306 AAAACCACAGAACATTCTGGTGACCA FGFR3 GCAGCTGGTGGAGGACCTGGACCGTGTCCTTACCGTGACGTCCACCGACG|TGATTGATGAGATTTATCGTGTGT 307 TGAGATATGTCAATTCTACCAGAGCC FGFR3 ACGACCTGCTGCCCCCGGCCCCACCCAGCAGTGGGGGCTCGCGGACGTGA|TTGATGAGATTTATCGTGTGTTGAG 308 ATATGTCAATTCTACCAGAGCCCCT FGFR3 CAGAGGCCCACCTTCAAGCAGCTGGTGGAGGACCTGGACCGTGTCCTTAC|CGTGACTGTTCTCAGGCGTGAAATT 309 TCTGAGCTTCGCACCAAAGTGCAAG FGFR3 CTTGCACTTTGGTGCGAAGCTCAGAAATTTCACGCCTGAGAACAGTCACG|GTAAGGACACGGTCCAGGTCCTCCA 310 CCAGCTGCTTGAAGGTGGG FGFR3 CCAACTGCACACACGACCTGTACATGATCATGCGGGAGTGCTGGCATGCC|GCCCCTCAACGAGCTCATGAAGTAC 311 TTCAAGAATTAAGGGATATATCCTC FGFR3 TGCACACACGACCTGTACATGATCATGCGGGAGTGCTGGCATGCCGCGCC|CTCCCAAGGTGATTGATGAGATTTA 312 TCGTGTGTTGAGATATGTCAATTCT FGFR3 AGGGGCTCTGGTAGAATTGACATATCTCAACACACGATAAATCTCATCAA|TCACGTCGGTGGACGTCACGGTAAG 313 GACACGGTCCAGGTCCTCCACCAGC FGFR3 CCCGCCAACTGCACACACGACCTGTACATGATCATGCGGGAGTGCTGGCA|ATGGAGTACTTTGATGAAAAGATTG 314 TTCCAATTTTAAAGAGGAAATTACC ZBTB17 GGCCTTCGTGAACGTGGGGGACCTGTCCAAGCACATCATCATTCACACTG|CATTAATAATAAGCTACAGCAGCCG 315 GAGGCAGCGGCCGGAGTGTTAGAAT KAZN CCCGCGCCGGGGTTCCCCGGGTCCGAGCGGATGGCGACTGCAGCCAGCCC|GGTCAGTGGGACAGCATGGAAGAAT 316 ACACCTGTATGATCCCTCGGGACAC EPHB2 CATGTGCAAAGCAGGCTTCGAGGCCGTTGAGAATGGCACCGTCTGCCGAG|TCCCGGCCAGTGTGCAGCTGCACAC 317 GGCGGTGGAGATGCACCACTGGTGC ERBB2 CCTGGCCACAGGTTGTCTTTTGCCCAGCAGACTGCCTGATGACCTTCAGG|GGGGCAACGTAGCCATCAGTCTCAG 318 AGGGCAGGGGTACTGTGGGGTCCTC ERBB2 CCCTCTGAGACTGATGGCTACGTTGCCCCCCTGACCTGCAGCCCCCAGCC|CTGCTGGGCAAAAGACAACCTGTGG 319 CCAGGGTCTGGAAGGGCCCTGGGAG JAK2 GACAAAGAATACTATAAAGTAAAAGAACCTGGTGAAAGTCCCATATTCTG|ATTGTGCGACGGGGAATGCCTCCCG 320 GAGGAGGAGGCGAAGTGGTTTTCTC JAK2 ACTTTTGAAGAATAATGGAAGATTACCAAGACCAGATGGATGCCCAGATG|AGGATAGAATTTTTGCGGCATTTGA 321 AGAGCTTTTTCCAGATTATGTTTAA CDKN2A CTACTGAGGAGCCAGCGTCTAGGGCAGCAGCCGCTTCCTAGAAGACCAGG|ATATTGAGGCAACAATGAACTCGGC 322 CCTGAATGAGCTACGGGAACTAGAA CDKN2A AGGCGGGGGCGCTGCCCAACGCACCGAATAGTTACGGTCGGAGGCCGATC|CAGGATATTGAGGCAACAATGAACT 323 CGGCCCTGAATGAGCTACGGGAACT FGFR3 GCAGCTGGTGGAGGACCTGGACCGTGTCCTTACCGTGACGTCCACCGACG|TAAAGGCGACACAGGAGGAGAACCG 324 GGAGCTGAGGAGCAGGTGTGAGGAG SLC7A2 CGCGGGCCCCCGACGCGCTGCAGCCGGCAGCCCACCGCCGCCTTCTTGGC|TGTGTTCCGGCCGCAGAGCACCGTC 325 TGCGTGAGGAGATCCTGGCCAAGTT RAF1 GGTGCGAGGGCAGCAGCCTCTACAAACACCTGCATGTCCAGGAGACCAAG|GAAAAGCCATGGAGACTTCAGCATC 326 CTCCTCCCAGCCTCAGGACAACAGT BRAF GGCCGGCGCCGGCGCCGGCGCCGCGGCCTCTTCGGCTGCGGACCCTGCCA|TTCCGGATATTGACTGTTCAAGTAC 327 TATTATGCTGGACAATATTGTGAGG TRIM24 ATTTGAGCTCACCAGTGGGAGGGTCTTATAATCTTCCCTCTCTTCCGGAT|GTGTGGAATATCAAACAAATGATTA 328 AGTTGACACAGGAACATATAGAGGC

TABLE 50 Pre-registration Gene approved (pre-approval) Phase III ABL1 imatinib mesilate N N bosutinib nilotinib ponatinib radotinib ALK crizotinib N N alectinib hydrochloride ceritinib BRAF vemurafenib N encorafenib pazopanib dabrafenib CDKN2A N N N CDK6 N palbociclib LEE-011 LY-2835219 EGFR panitumumab N neratinib cetuximab XL-647 erlotinib rindopepimut nepidermin necitumumab gefitinib dacomitinib nimotuzumab rociletinib vandetanib AZD-9291 lapatinib afatinib icotinib Gene Phase II Phase I Preclinical ABL1 bafetinib rebastinib ON-044580 ABL-001 SUN-K706 ALK AP-26113 CEP-37440 AZD-3463 X-396 EBI-215 ARN-5032 PF-06463922 DLX-521 TSR-011 entrectinib BRAF RAF-265 ARQ-761 Braf inhibitors, Sareum XL-281 ARQ-736 B-Raf and c-RAF dual AB-024 BeiGene-283 inhibitor, Redx Pharma PLX-8394 DP-4978 PLX-8394 DP-4978 TL-241, B-Raf kinase inhibitors, Array BioPharma-1 EN-3352 EBI-907 PI3K/BET bromodomain inhibitors SignalRx UAI-201 Nanolipolee-007 ASN-003 CDKN2A P16_37-63 N N CDK6 alvocidib GZ38-1 CDK4/6 inhibitors, G1 Therapeutics capridine Beta, AV Therapeutics G1T38-1 G1T30-1 EGFR dovitinib JNJ-26483327 CUDC-101 varlitinib MM-151 Sym-013 marizomib AL-6802 STI-A020X futuximab ABT-806 SCT-200 S-222611 epitinib pirotinib AP-26113 allitinib PF-06459988 antroquinonol theliatinib NT-004 GT-MAB 5.2-GEX ErbituxEDVsPac larotinib poziotinib AMG-595 KL-ON113 duligotuzumab IMGN-289 JNJ-61186372 LY-3016859 pyrotinib IRAD-425 ABT-414 TAS-121 HL-176 KD-020 chimeric antigen receptor FV-225 ASP-8273 T-cell therapy, Epidermal growth doxorubicin loaded EGFRVIII, Novartis factor, USV Limited EnGeneIC delivery GC-1118A EM1-mAB vehicles LY-3164530 EGFRvIII/CD3 EGF-816 AZD-3759 CUDC-101 EGFRvII CAR, miR-16, EDV nanocells, CTX-023 Kite Pharma EnGeneIC BPI-4039 BPI-0403 bi-specific antibodies, Zyngenia Avid Biologics-2 Avid Biologics-1 anticancer gene therapy, Gradalis anti-EGFR MAbs, Kadmon anti-EGFR biosimilar, Mabion AFM-21 Pre-registration Gene approved (pre-approval) Phase III ERBB2 trastuzumab N neratinib trastuzumab emtansine XL-647 pertuzumab; lapatinib dacomitinib ditosylate nelipepimut-S catumaxomab trastuzumab afatinib (Celltrion, Biocad, trastuzumab, Enhanze Biocon, Synthon, Harvest Moon, Aryogen) doxorubicin FGFR2 ponatinib lenvatinib N palifermin FGFR3 ponatinib lenvatinib N levetiracetam in masitinib sodium chloride, HQ Specialty Pharma JAK2 ruxolitinib N momelotinib pacritinib baricitinib MTOR everolimus ridaforolimus TCD-10023 temsirolimus voxtalisib sirolimus nab-rapamycin zotarolimus apitolisib biolimus gedatolisib umirolimus NTRK1 N N MIM-D3 RAF1 sorafenib N N TERT N N GV-1001 Gene Phase II Phase I Preclinical ERBB2 lapuleucel-T Her-VAXX Lovaxin B AVX-901 VM-206 TH-1, Algeta AE-37 ARRY-380 trastuzumab-antibody BMS-690514 JNJ-26483327 conjugates, Synthon MVA-BN-HER2 S-222611 CUDC-101 varlitinib doxorubicin (Merrimack) Her-2/neu Stradobody,Gliknik MM-111 cipatinib ARX-788 AC-480; ovarian TrasGEX Etbx-021 cancer trastuzumab (Hanwha SN-34003 vaccine (Generex) Chemical); IBI-302 TrasGEX trastuzumab (Pfizer) NT-004 ICT-140 margetuximab IDN-6439 ONS-1050 poziotinib AVX-901 Sym-013 PR-610 anti-HER2 X anti-CD3 KD-020 (Emergent Biosolutions) FGFR2 EN MD-2076 JNJ-42756493 FPA-144 lucitanib S-49076 keratinocyte growth AZD-4547 FF-284 factor, Nanogen BGJ-398 BAY-1179470 LY-2874455 BAY-1163877 ASP-5878 FGFR3 dovitinib lactate JNJ-42756493 N ENMD-2076 S-49076 AZD-4547 FF284 BGJ-398 BAY-1163877 LY-2874455 ASP-5878 B-701 JAK2 BMS-911543 AC-430 ON-044580 NS-018 SB-1317 TP-0413 PF-06263276 VR-588 NMS-P953 MTOR quinacrine P-7170 nPT-MTOR XL-765 CBLC-137 STP-503 dactolisib AZD-2014 X-480 PKI-587 CC-115 ABTL-0812 PF-04691502 PWT-33957 X-414; CC214 INK-128 DS-7423 HMPL-518 AZD-2014 GDC-0084 PQR-401 CC-223 DS-3078 mTOR inhibitor/PI3 kinase LY-3023414 inhibitor, Lilly-1 PI3 kinase/mTOR PIM/PI3k/mTOR inhibitors, inhibitor, Lilly Inflection Biosciences SF-1126 LOR-220 SB-2343 NV-128 omipalisib SPR-965 ABTL-0812 IBL-301 PQR-309 PQR-316 DCBCI-0901 PQR-311 TAM-01 NTRK1 milciclib maleate BXL-1H5 NMS-P626 CT-327 PLX-7486 CT-340 NMS-E628 LOXO-101 FX-007 PSR-011 DS-6051b CRB-0089 pan-TRK (Merck) RAF1 iCo-007 RO-5126766 HM-95573 XL-281 MLN-2480 TAK-632 DP-4978 UAI-201 pan-RAF Novartis and Celator TERT VX-001 TeloB-Vax telomerase vaccine, Geron GX-301 hTERT DNA vaccine, lnovio INVAC-1 AST-VAC2 

What is claimed:
 1. A composition reaction mixture comprising a target nucleic acid and a set of primers that specifically hybridize to the target nucleic acid to flank a gene fusion, a recombinant thermostable polymerase, and dNTPs, wherein the set of primers specifically amplify a fusion between the KANK2 gene and the ALK gene comprising SEQ ID NO: 272, and wherein each primer is 15 to 40 nucleotides long and a deoxyribonucleic acid sequence comprising at least one deoxyuridine (dU) residue in place of a deoxythymidine residue.
 2. The composition of claim 1, additionally comprising one or more additional set of primers that specifically hybridize to one or more additional target nucleic acids to flank a gene fusion, wherein the additional set(s) of primers specifically amplify at least one fusion of two genes comprising one or more sequences selected from SEQ ID NOs: 1-328.
 3. A kit comprising components: i. a set of primers that specifically hybridize to a target nucleic acid to flank a gene fusion, wherein the set of primers specifically amplify a fusion between the KANK2 gene and the ALK gene comprising SEQ ID NO: 272, and wherein each primer is 15 to 40 nucleotides long and a deoxyribonucleic acid sequence comprising at least one deoxyuridine (dU) residue in place of a deoxythymidine residue, ii. a recombinant thermostable polymerase, and iii. a set of dNTPs.
 4. The kit of claim 3, additionally comprising one or more additional set of primers that specifically hybridize to a target nucleic acid to flank a gene fusion that specifically amplify at least one fusion of two genes comprising one or more sequences selected from SEQ ID NOs: 1-328.
 5. The composition of claim 2, wherein the set of primers specifically hybridize to at least one nucleic acid comprising a break point from sequences selected from SEQ ID NOs: 1-328. 